JP2015131828A - Compositions and methods for modulation of smn2 splicing in subject - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide compounds, compositions containing the same and methods using the same for modulating splicing of survival motor neuron gene 2 (SMN2) mRNA in a subject.SOLUTION: An antisense oligonucleotide compound for a subject is complementary to intron 7 of a nucleic acid encoding human SMN2 pre-mRNA and includes a modified sugar component or a phosphorothioate internucleoside linkage. Also provided are a composition containing the same and a method comprising administering the same into the cerebrospinal fluid.

Description

SEQUENCE LISTING
This application is filed with Sequence Listing in electronic format. This Sequence Listing was created on June 17, 2010 and is provided as a file titled “20100617_CORE0086WOSEQ.txt” with a size of 5 Kb. This information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

Newly synthesized eukaryotic mRNA molecules, known as primary transcripts or pre-mRNAs, are processed prior to translation. Pre-mRNA processing includes the addition of a 5 ′ methylation cap and the addition of a roughly 200-250 base poly (A) tail transcript to the 3 ′ end. Processing of mRNA from pre-mRNA is also often associated with pre-mRNA splicing, which occurs upon 90-95% maturation of mammalian mRNA. An intron (or intervening sequence) is a region of pre-mRNA (or the DNA that encodes it) that is not included in the coding sequence of the mature mRNA. Exons are regions of primary transcript that remain in the mature mRNA. Exons are spliced together to form the mature mRNA sequence. Splice junctions also have a 5 'side of the junction often referred to as a "5' splicing site" or "splicing donor site" and a 3 'side of the junction often referred to as a "3' splicing site" or "splicing acceptor site" Referred to as the splicing site. Upon splicing, the 3 ′ end of the upstream exon is joined with the 5 ′ end of the downstream exon. Thus, the unspliced pre-mRNA has an exon / intron junction at the 5 ′ end of the intron and an intron / exon junction at the 3 ′ end of the intron. After the intron is removed, the exon is adjacent to a part often referred to as an exon / exon junction or boundary in the mature mRNA. An ambiguous splicing site is a splicing site that is used less often but can be used when the normal splicing site is blocked or unavailable. Alternative splicing is defined as splicing with different combinations of exons, but often results in multiple mRNA transcripts from a single gene.

Up to 50% of human genetic diseases resulting from point mutations result in abnormal pre-mRNA processing. Such point mutations can destroy existing splicing sites or form new splicing sites, resulting in mRNA transcripts containing different combinations of exons, or mRNAs containing deletions in exons. This produces a transcript. Point mutations can also result in subambiguous activation of the splicing site or can disrupt regulatory cis elements (ie, splicing enhancers or silencers) (Cartegni et al., Nat. Rev. Genet ., 2002, 3, 285-298; Drawczak et al., Hum. Genet.,
1992, 90, 41-54). To direct splicing to obtain the desired splicing product, antisense oligonucleotides were used to target mutations that cause abnormal splicing in several genetic diseases (Kole, Acta Biochimica Polonica, 1997, 44 , 231-238).

Antisense compounds are also used to alter the proportion of naturally occurring alternative splicing variants such as the long and short forms of Bcl-x pre-mRNA (US Patent 6,172,216; US Patent 6,214,986; Taylor et al. , Nat. Biotechnol. 1999, 17, 1097-1100), or forced skipping of specific exons containing an immature stop codon (Wilton e
t al., Neuromuscul. Disord., 1999, 9, 330-338). US Patent 5,627,274 and WO 94/26887 describe compositions and methods for resisting abnormal splicing in mutation-containing pre-mRNA molecules containing mutations using antisense oligonucleotides that do not activate RNAse H. Disclose.

Proximal spinal muscular atrophy (SMA) is an inherited neurodegenerative disease characterized by the loss of spinal motor neurons. SMA is an early-onset autosomal recessive disorder and the leading cause of death among current infants. The severity of SMA varies from patient to patient and is therefore classified into these types. Type I SMA is the most severe form that develops at birth or within 6 months and is fatal within 2 years. Children with type I SMA cannot sit or walk. Type II SMA is an intermediate type that allows a patient to sit but cannot stand or walk. Patients with type III SMA, a chronic form of disease, typically develop SMA after 18 months of age (Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536).

The molecular basis of SMA arises from the loss of both copies of survival motor neuron gene 1 (SMN1). This SMN1 is also known as SMN Telomeric, a protein that is part of a multi-protein complex thought to be involved in snRNP biogenesis and recycling. SMN2, an almost identical gene, also known as SMN Centromeric, is present in a replicated region on chromosome 5q13 and modifies disease severity. Expression of the normal SMN1 gene slightly results in the expression of survival motor neuron (SMN) protein. SMN1 and SMN2 have the potential to encode the same protein, but SMN2 contains a translationally silent mutation at position +6 of exon 7, which is inefficient in exon 7 of the SMN2 transcript Contains. Thus, the dominant form of SMN2 is a truncated form lacking exon 7, which is unstable and inactive (Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384 ). SMN2 gene expression is approximately 10-20% SMN protein and 80-90
% Of unstable / non-functional SMN delta 7 protein is produced. SMN proteins perform well-established functions in spliceosome assembly and may mediate mRNA trafficking in neuronal axons and nerve endings.

Antisense technology is an effective means for modulating the expression of one or more specific gene products, including alternative splicing products, and is characterized in numerous therapeutic, diagnostic, and research applications Useful. The principle behind antisense technology is that an antisense compound that hybridizes to a target nucleic acid modifies gene expression activity such as transcription, splicing or translation through one of many antisense technologies It is. The sequence specificity of antisense compounds makes them extremely attractive as tools for target confirmation and gene functionalization, and as therapeutics to selectively modify the expression of genes associated with disease. Yes.

  Certain antisense compounds complementary to SMN2 are known in the art. For example, WO 2007/002390; US 61 / 168,885; Hua et al., American J. of Human Genetics (April 2008) 82, 1-15; Singh et al., RNA Bio. 6: 3, 1-10 (2009 See). Certain antisense compounds and methods disclosed herein have desirable characteristics compared to such compounds and methods in the art. Chimeric peptide nucleic acid molecules designed to modify splicing of SMN2 have been described (WO 02/38738; Cartegni and Krainer, Nat. Struct. Biol., 2003, 10, 120-125).

U.S. Patent 6,172,216 U.S. Patent 6,214,986 U.S. Patent 5,627,274 WO 94/26887 WO 2007/002390 US 61 / 168,885 WO 02/38738

Cartegni et al., Nat. Rev. Genet., 2002, 3, 285-298 Drawczak et al., Hum. Genet., 1992, 90, 41-54 Kole, Acta Biochimica Polonica, 1997, 44, 231-238 Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100 Wilton et al., Neuromuscul. Disord., 1999, 9, 330-338 Lefebvre et al., Hum. Mol. Genet., 1998, 7, 1531-1536 Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384 Hua et al., American J. of Human Genetics (April 2008) 82, 1-15 Singh et al., RNA Bio. 6: 3, 1-10 (2009) Cartegni and Krainer, Nat.Struct. Biol., 2003, 10, 120-125

In certain embodiments, the invention comprises a method comprising administering to a subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 pre-mRNA. Wherein the antisense compound is administered in cerebrospinal fluid. In certain embodiments, administration occurs in the subarachnoid space. In certain embodiments, administration is in cerebrospinal fluid within the brain. In certain embodiments, administration includes bolus injection. In certain embodiments, administration includes infusion using a delivery pump.

In certain embodiments, the antisense compound is administered at a dose of 0.01 to 10 milligrams of antisense compound per kg body weight of the subject. In certain embodiments, the dose is 0.01 to 10 milligrams of antisense compound per kg body weight of the subject. In certain embodiments, the dose is 0.01 to 5 milligrams of antisense compound per kg body weight of the subject. In certain embodiments, the dose is 0.05 to 1 milligram of antisense compound per kg body weight of the subject. In certain embodiments, the dose is 0.01 to 0.5 milligrams of antisense compound per kg body weight of the subject. In certain embodiments, the dose is 0.05 to 0.5 milligrams of antisense compound per kg body weight of the subject.

In certain embodiments, the dose is administered daily. In certain embodiments, the dose is administered once a week. In certain embodiments, the antisense compound is administered continuously and the dosage is the amount administered per day. In certain embodiments, the method comprises administering at least one induction dose during the induction phase and administering at least one maintenance dose during the maintenance phase. In certain embodiments, the induction phase is 0.05 to 5.0 milligrams of antisense compound per kg body weight of the subject. In certain embodiments, the maintenance dose is 0.01 to 1.0 milligrams of antisense compound per kg body weight of the subject. In certain embodiments, the duration of the induction phase is at least 1 week. In certain embodiments, the duration of the maintenance phase is at least 1 week. In certain embodiments, each induction administration and each maintenance administration comprises a single injection. In certain embodiments, each induction administration and each maintenance administration independently comprises two or more injections. In certain embodiments, the antisense compound is administered at least twice over a treatment period of at least 1 week. In certain embodiments, the treatment period is at least 1 month. In certain embodiments,
The treatment period is at least 2 months. In certain embodiments, the treatment period is at least 4 months. In certain embodiments, the induction phase is administered by one or more bolus injections and the maintenance dose is administered by an infusion pump.

  In certain embodiments, the method includes assessing the tolerability and / or effectiveness of the antisense compound. In certain embodiments, the dosage or frequency of administration of the antisense compound is reduced according to indications that do not allow administration of the antisense compound. In certain embodiments, the dosage or frequency of administration of the antisense compound is maintained or reduced according to the indication for which administration of the antisense compound is effective. In certain embodiments, the dosage of the antisense compound is increased according to an indication for which administration of the antisense compound is not effective. In certain embodiments, the frequency of administration of the antisense compound is reduced according to the indication for which administration of the antisense compound is effective. In certain embodiments, the frequency of administration of the antisense compound is increased according to indications for which administration of the antisense compound is not effective.

In certain embodiments, the method includes co-administering an antisense compound and at least one other treatment. In certain embodiments, the antisense compound and at least one
Two other treatments are co-administered simultaneously. In certain embodiments, the antisense compound is administered prior to administration of at least one other treatment. In certain embodiments, the antisense compound is administered after administration of at least one other treatment. In certain embodiments, at least one other treatment is valproic acid, riluzole, hydroxyurea,
And administering one or more of butyrate. In certain embodiments, at least one other treatment comprises administering trichostatin-A. In certain embodiments, at least one other treatment includes administering a stem cell. In certain embodiments, the at least one other treatment is gene therapy. In certain embodiments, gene therapy is administered to the CSF and the antisense compound is administered systemically. In certain embodiments, gene therapy is administered to CSF and antisense compounds are administered systemically and CSF. In certain embodiments, the present invention provides a therapeutic formulation in which the antisense compound is first administered CSF and systemically, then gene therapy is administered to the CSF, and the antisense compound is administered systemically. In certain such embodiments, the subject is an infant at the time of initial treatment. In certain such embodiments, the subject is less than 2 years old. In certain embodiments, the antisense compound is administered to the subject's CNS until the subject is of sufficient age for gene therapy. In certain such embodiments, the antisense compound is administered systemically throughout.

  In certain embodiments, the antisense compound is about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 5 mg / ml, about 10 mg / ml. Dosing at a concentration of ml, about 50 mg / ml, or about 100 mg / ml.

  In certain embodiments, the content of exon 7 of SMN2 mRNA in the subject's motor neurons is increased. In certain embodiments, the exon 7 amino acid content of the SMN2 polypeptide in the subject's motor neurons is increased.

In certain embodiments, the invention administers to a subject an antisense compound comprising an antisense oligonucleotide that is complementary to intron 7 of a nucleic acid encoding human SMN2, and thereby the subject Increasing the content of exon 7 of SMN2 mRNA in a motor neuron of the subject, the method comprising increasing the content of exon 7 of SMN2 mRNA in a motor neuron of a subject.

In certain embodiments, the invention administers to a subject an antisense compound comprising an antisense oligonucleotide that is complementary to intron 7 of a nucleic acid encoding human SMN2, and thereby There is provided a method of increasing the amino acid content of exon 7 in an SMN2 polypeptide in a motor neuron of a subject, comprising increasing the content of exon 7 amino acid in the SMN2 polypeptide in the motor neuron.

  In certain embodiments, the subject has SMA. In certain embodiments, the subject has type I SMA. In certain embodiments, the subject has type II SMA. In certain embodiments, the subject has type III SMA.

In certain embodiments, the first dose is administered into the uterus. In certain embodiments, the first dose is administered before completing the formation of the blood brain barrier formation. In certain embodiments, the first dose is administered within one week of life of the subject. In certain embodiments, the first dose is administered within one month of life of the subject. In certain embodiments, the first dose is administered within 3 months of life of the subject. In certain embodiments, the first dose is administered within 6 months of life of the subject. In certain embodiments, the first dose is administered when the subject is 1-2 years old. In certain embodiments, the first dose is administered when the subject is 1-15 years old. In certain embodiments, the first
Is administered when the subject is 15 years of age or older.

  In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

In certain embodiments, the method includes identifying a subject having SMA. In certain embodiments, a subject is identified by measuring the electrical activity of one or more subject's muscles. In certain embodiments, the subject is identified by genetic testing to determine whether the subject has a mutation in the subject's SMN1 gene. In certain embodiments, the subject is identified by muscle biopsy.

In certain embodiments, administering an antisense compound results in an increase in the amount of SMN2 mRNA having at least 10% exon 7. In certain embodiments, the increase in the amount of SMN2 mRNA having exon 7 is at least 20%. In certain embodiments, the increase in the amount of SMN2 mRNA having exon 7 is at least 50%. In certain embodiments, the amount of SMN2 mRNA having exon 7 is at least 70%.

In certain embodiments, administering an antisense compound results in an increase in the amount of SMN2 polypeptide having at least 10% exon 7 amino acids. In certain embodiments, the increase in the amount of SMN2 polypeptide having an amino acid of exon 7 is at least 20%. In certain embodiments, the increase in the amount of SMN2 polypeptide having an amino acid of exon 7 is at least 50%. In certain embodiments, the increase in the amount of SMN2 polypeptide having an amino acid of exon 7 is at least 70%.

In certain embodiments, administering an antisense compound ameliorates at least one symptom of SMA in the subject. In certain embodiments, administration of an antisense compound results in an improvement in the subject's motor function. In certain embodiments, administering an antisense compound results in delaying or reducing the loss of motor function in the subject. In certain embodiments, administration of an antisense compound results in improved respiratory function. In certain embodiments, administration of an antisense compound results in improved survival.

In certain embodiments, at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety. In certain embodiments, the at least one modified sugar component comprises a 2′-methoxyethyl sugar component. In certain embodiments, essentially each nucleoside of the antisense oligonucleotide comprises a modified sugar moiety. In certain embodiments, all nucleosides that include a modified sugar moiety include the same sugar modification. In certain embodiments, each modified sugar component comprises a 2′-methoxyethyl sugar component. In certain embodiments, each nucleoside of the antisense oligonucleotide comprises a modified sugar moiety. In certain embodiments, all of the nucleosides contain the same sugar modification. In certain embodiments, each modified sugar component comprises a 2′-methoxyethyl sugar component. In certain embodiments, at least one internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

  In certain embodiments, the antisense oligonucleotide consists of 10-25 linked nucleosides. In certain embodiments, the antisense oligonucleotide consists of 12-22 linked nucleosides. In certain embodiments, the antisense oligonucleotide consists of 15-20 linked nucleosides. In certain embodiments, the antisense oligonucleotide consists of 18 linked nucleosides.

  In certain embodiments, the antisense oligonucleotide is at least 90% complementary to a nucleic acid encoding human SMN2. In certain embodiments, the antisense oligonucleotide is completely complementary to a nucleic acid encoding human SMN2. In a particular embodiment, the oligonucleotide has a nucleobase sequence comprising at least 10 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising at least 15 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 1. In certain embodiments, the oligonucleotide has a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1. In a particular embodiment, the oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence SEQ ID NO: 1.

  In certain embodiments, the antisense compound comprises a conjugated group or a terminal group.

  In certain embodiments, the antisense compound consists of an antisense oligonucleotide.

  In certain embodiments, the antisense compound is also administered systemically. In certain embodiments, systemic administration is by intravenous or intraperitoneal injection. In certain embodiments, systemic administration and administration to the central nervous system occur simultaneously. In certain embodiments, systemic administration and administration to the central nervous system are performed at different times.

In certain embodiments, the present invention provides systemic administration of antisense compounds alone or with delivery into CSF. In certain embodiments, the pharmaceutical composition is administered systemically. In certain embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments, the pharmaceutical composition is administered by intramuscular injection.

In certain embodiments, the pharmaceutical composition is administered both directly (eg, IT and / or ICV injection and / or infusion) and systemically to the CSF.

In certain embodiments, the invention comprises, for a subject having at least one symptom associated with SMA, consisting of 15-20 linked nucleosides and is 100% complementary to SEQ ID NO. 7 over its entire length And a method of administering at least one dose of an antisense compound comprising an oligonucleotide having a typical nucleobase sequence, wherein each nucleoside is a 2'-MOE modified nucleoside; and at least one dose of CSF Administered against 0.
It is in the range of 1 mg / kg to 5 mg / kg. In certain such embodiments, the dose is 0.5 mg / kg to 2
It is in the range of mg / kg. In certain embodiments, at least one dose is administered by bolus injection. In certain such embodiments, the dose is administered by bolus subarachnoid injection. In certain embodiments, at least one second dose is administered. In certain such embodiments, the second dose is administered at least 2 weeks after the first dose. In certain embodiments, the second dose is administered at least 4 weeks after the first dose. In certain embodiments,
The second dose is administered at least 8 weeks after the first dose. In certain embodiments, the second dose is administered at least 12 weeks after the first dose. In certain embodiments, the second dose is administered at least 16 weeks after the first dose. In certain embodiments, the second dose is administered at least 20 weeks after the first dose. In certain embodiments, the subject is 2 years or younger at the time of the first dose. In certain embodiments, the subject ranges from 2 to 15 years old. In certain embodiments, the subject ranges from 15 to 30 years. In certain embodiments, the subject is 30 years or older. In certain embodiments, at least one symptom associated with SMA is reduced or slowed. In a particular embodiment, the oligonucleotide is ISIS396443.

In certain embodiments, the invention comprises, for a subject having at least one symptom associated with SMA, consisting of 15-20 linked nucleosides and is 100% complementary to SEQ ID NO. 7 over its entire length A method is provided for administering at least one dose of an antisense compound comprising an oligonucleotide having a typical nucleobase sequence, wherein each nucleoside is a 2'-MOE modified nucleoside; and at least one dose is administered systemically Administer. In certain such embodiments, at least one dose is administered by bolus injection. In certain such embodiments, the dose is administered by bolus subcutaneous injection. In certain embodiments, the dosage ranges from 0.5 mg / kg to 50 mg / kg. In certain embodiments, the dose ranges from 1 mg / kg to 10 mg / kg. In certain embodiments, the dose ranges from 1 mg / kg to 5 mg / kg. In certain embodiments, the dose ranges from 0.5 mg / kg to 1 mg / kg. In certain embodiments, at least one second dose is administered. In certain such embodiments, the second dose is administered at least 2 weeks after the first dose. In certain embodiments, the second dose is administered at least 4 weeks after the first dose. In certain embodiments, the second dose is administered at least 8 weeks after the first dose. In certain embodiments, the second dose is administered at least 12 weeks after the first dose. In certain embodiments, the second dose is administered at least 16 weeks after the first dose. In certain embodiments, the second dose is administered at least 20 weeks after the first dose. In certain embodiments, the subject is 2 years of age or younger at the first dose. In certain embodiments, the subject ranges from 2 to 15 years old. In certain embodiments, the subject ranges from 15 to 30 years. In certain embodiments, the subject is 30 years or older. In certain embodiments, at least one symptom associated with SMA is reduced or slowed. In a particular embodiment, the oligonucleotide is ISIS396443.

In certain embodiments, the invention comprises, for a subject having at least one symptom associated with SMA, consisting of 15-20 linked nucleosides and 100% complementary to SEQ ID NO. 7 over its entire length Administering at least one dose to the CSF and at least one systemic dose of an antisense compound comprising an oligonucleotide having a specific nucleobase sequence, wherein each nucleoside is a 2'-MOE modified nucleoside It is. In certain such embodiments, the CSF dose ranges from 0.1 mg / kg to 5 mg / kg. In certain embodiments, the systemic dose ranges from 0.5 mg / kg to 50 mg / kg. In certain embodiments, at least one CSF dose is administered by bolus injection. In certain such embodiments, at least one CSF dose is administered by bolus subarachnoid injection. In certain embodiments, at least one systemic dose is administered by bolus injection. In certain such embodiments, at least one systemic dose is administered by subcutaneous injection. In certain embodiments, the CSF dose and the systemic dose are administered simultaneously. In certain embodiments, the CSF dose and the systemic dose are administered at different times. In certain embodiments, the subject is 2 years or younger at the time of the first dose. In certain embodiments, the subject ranges from 2 to 15 years. In certain embodiments, the subject ranges from 15 to 30 years. In certain embodiments, the subject is 30 years or older. In certain embodiments, at least one symptom associated with SMA is reduced or slowed. In a particular embodiment, the oligonucleotide is ISIS396443.

In certain embodiments, the invention comprises, for a subject having at least one symptom associated with SMA, consisting of 15-20 linked nucleosides and 100% complementary to SEQ ID NO. 7 over its entire length And a method of administering at least one systemic dose of an antisense compound comprising an oligonucleotide having a typical nucleobase sequence, wherein each nucleoside is a 2'-MOE modified nucleoside; and at least one dose of Administer gene therapy agent. In certain embodiments, the systemic dose is in the range of 0.5 mg / kg to 50 mg / kg. In certain embodiments, at least one systemic dose is administered by bolus injection. In certain such embodiments, at least one systemic dose is administered by subcutaneous injection. In certain embodiments, the systemic dose and the gene therapy agent are administered simultaneously. In certain embodiments, the systemic dose and the gene therapy agent are administered at different times. In certain embodiments, the gene therapy agent is administered to CSF. In certain such embodiments, the gene therapy agent is administered by subarachnoid injection and / or infusion. In certain such embodiments, the gene therapy agent is administered by intracerebroventricular injection and / or infusion. In certain embodiments, the subject is 2 years or younger at the time of the first dose. In certain embodiments, the subject ranges from 2 to 15 years. In certain embodiments, the subject ranges from 15 to 30 years. In certain embodiments, the subject is 30 years or older. In certain embodiments, at least one symptom associated with SMA is reduced or slowed. In a particular embodiment, the oligonucleotide is ISIS396443.

  In certain embodiments, the invention provides a method of selecting a subject having at least one symptom associated with SMA and administering an antisense compound according to any of the methods described above. In certain such embodiments, at least one symptom of SMA is assessed after administration. In certain such embodiments, at least one symptom of SMA is ameliorated. In certain such embodiments, at least one symptom of SMA progresses slowly compared to a subject who does not progress or who has not received an antisense compound.

In certain embodiments, the present invention provides an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 for use in any of the methods described above. In certain embodiments, the present invention provides such compounds for use in treating a disease or condition associated with survival motor neuron 1 (SMN1).

In certain embodiments, the invention provides an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2, in the manufacture of a medicament for use in any of the methods described above. Provide use. In certain embodiments, the medicament is for treating a disease or condition associated with survival motor neuron 1 (SMN1).

FIG. 1 shows the results from the duration-of-action study examined in Example 4. In this example, the percentage of SMN2 with exon 7 (y-axis) was evaluated at 0, 2, 4, 6, and 8 weeks (x-axis) after the end of 7 days of treatment. “0” week samples were taken one day after the end of treatment. Controls represent mice treated with saline solution. There was no difference in% inclusion among control saline treated mice at different time points from 0-6 months. FIG. 2 shows the results from the duration-of-action study examined in Example 4. In this example, the proportion of SMN2 with exon 7 was evaluated at 0, 0.5, 1, 2, 5, and 6 months after the end of 7 days of treatment. A “0” month sample was taken one day after the end of treatment. CON indicates mice treated with saline solution. There was no difference in% inclusion among control saline treated mice at different time points from 0-6 months. FIG. 3 shows the results from the experiment studied in Example 6, in which the effect of embryonic administration of ISIS 396443 on the tail area was measured in Taiwan strains of SMA mice. FIG. 3A shows the first such experiment, and FIG. 3B shows the results of repeated experiments testing the different concentrations of antisense compounds shown and results including data on normal mice for comparison. . FIG. 4 shows the results from the Western blot examined in Example 7. The Y axis is the percentage of SMN in various samples including exon 7. FIGS. 5 and 6 show the results derived from the experiment studied in Example 7. FIG. Numerous evaluations of SMA mice (Taiwan strain) were performed following treatment with either antisense compounds or control oligonucleotides. FIGS. 5 and 6 show the results derived from the experiment studied in Example 7. FIG. Numerous evaluations of SMA mice (Taiwan strain) were performed following treatment with either antisense compounds or control oligonucleotides. FIG. 7 shows the survival curve from the experiment studied in Example 7. FIG. 8 shows the results from assessment of the number of motor neurons in different sites of the spinal cord after treatment with antisense compounds or control oligonucleotides as discussed in Example 7. FIG. 9 shows the results from evaluation of complete SMN RNA (including exon 7) in animals treated with the antisense studied in Example 7, as discussed in Example 7. FIG. 10 shows the survival curve from the experiment studied in Example 7. In this experiment, animals were (1) untreated; (2) at birth, a single dose of antisense compound (Day P0); or (3) the first dose on P0 and the second dose on day 21 ( P21); FIG. 11 shows the survival curve from the experiment described in Example 7, comparing animals receiving the second dose with animals receiving only the first dose. FIG. 12 shows the results from the experiment studied in Example 9. In this experiment, antisense compounds were administered to monkeys by infusion, and compound concentrations were evaluated in different tissues after 96 hours. FIG. 13 shows the survival curve from the experiment studied in Example 12. In this experiment, different doses of antisense compounds were administered to SMA mice by subcutaneous injection.

DETAILED DESCRIPTION OF THE INVENTION Both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the invention as recited in the claims. You should understand that. In this specification, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” and other forms such as “included” is not limiting. Similarly, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or parts of documents, cited in this application, including but not limited to patents, patent applications, literature, books, technical books, etc., are incorporated in their entirety for any purpose. Is expressly incorporated by reference.

I. Definitions Unless otherwise defined, the terminology used in connection with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein, and analytical chemistry, synthetic organic Chemical and medical and medicinal chemistry procedures and techniques are well known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. Certain such techniques and procedures can be found in the following literature (eg, “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington DC, 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and “Antisense Drug Technology, Principles, Strategies, and Applications”
Edited by Stanley T. Crooke, CRC Press , Boca Raton, Florida;. And Sambrook et al, "Molecular Cloning, A laboratory Manual," 2 nd Edition, Cold Spring Harbor
Laboratory Press, 1989), which are incorporated by reference for any purpose.
Where allowed, all patents, applications, published applications and other publications and other data mentioned throughout the disclosure herein are incorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the following meanings:
“Nucleoside” means a compound comprising a heterocyclic base moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides, modified nucleosides, and nucleosides and / or sugar groups with mimetic bases. The nucleoside may be modified with any of a variety of substituents.

  “Sugar component” means a natural sugar, or a modified sugar or sugar substitute.

  “Natural sugar” refers to the ribofuranose component of DNA (2′-H) or RNA (2′-OH).

“Modified sugar” means a ribofuranose moiety that contains at least one substituent other than a substituent of a natural sugar.

“Sugar substitute” means a structure other than a ribofuranose ring that can replace the sugar of a nucleoside. Examples of sugar substitutes include, but are not limited to, ring-opened systems, 6-membered rings, sugars in which oxygen is replaced by, for example, sulfur or nitrogen. For example, sugar substitutes include, but are not limited to, morpholino and 4′-thio-containing sugars.

  “Nucleobase” means the heterocyclic base portion of a nucleoside. Nucleobases may be naturally occurring or modified. In certain embodiments, a nucleobase may include any atom or group that can hydrogen bond to a nucleobase of another nucleic acid.

“Nucleotide” means a nucleoside containing a phosphate linking group. As used herein, a nucleoside includes a nucleotide.

A “modified nucleoside” is a nucleoside that contains at least one modification compared to a naturally occurring RNA or DNA nucleoside. Such modifications may be present on the sugar component and / or the nucleobase.

  “Bicyclic nucleoside” or “BNA” means a nucleoside in which the sugar component of the nucleoside includes a bridge connecting the two carbon atoms of the sugar ring, thereby forming a bicyclic sugar component.

  “4′-2 ′ bicyclic nucleoside” means a bicyclic nucleoside containing a furanose ring containing a bridge connecting the two carbon atoms of the furanose ring, the 2 ′ and 4 ′ carbon atoms of the sugar ring And

“2′-Modified” or “2′-Substituted” means a nucleoside containing a sugar that contains a substituent at the 2 ′ position other than H or OH.

“2′-OMe” or “2′-OCH ₃ ” or “2′-O-methyl” represents —OCH _{3 at} the 2 ′ position of the sugar ring, respectively.
A nucleoside containing a sugar containing a group is meant.

“MOE” or “2′-MOE” or “2′-OCH ₂ CH ₂ OCH ₃ ” or “2′-O-methoxyethyl” represents an —OCH ₂ CH ₂ OCH ₃ group at the 2 ′ position of the sugar ring, respectively. Means a nucleoside containing a sugar containing

“Oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more nucleosides are modified. In certain embodiments, the oligonucleotide comprises one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

  “Oligonucleoside” means an oligonucleotide that contains no phosphorus atom anywhere in the internucleoside linkage. As used herein, an oligonucleotide includes an oligonucleoside.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage.

  “Internucleoside linkage” means a covalent linkage between adjacent nucleosides of an oligonucleotide.

  “Naturally occurring internucleoside linkage” means a 3′-5 ′ phosphodiester linkage.

  “Modified internucleoside linkage” means any internucleoside linkage other than naturally occurring internucleoside linkages.

  “Oligomer compound” means a compound comprising an oligonucleotide. In certain embodiments, the oligomeric compound consists of an oligonucleotide. In certain embodiments, the oligomeric compound further comprises one or more conjugates and / or end groups.

“Antisense compound means an oligomeric compound, at least part of which is at least partially complementary to the target nucleic acid, wherein such hybridization is:
Produces at least one antisense activity.

  “Antisense oligonucleotide” means an antisense compound in which the oligomeric compound consists of oligonucleotides.

“Antisense activity” refers to any detectable and / or measurable effect that can contribute to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, such antisense activity is an increase or decrease in the amount of nucleic acid or protein. In certain embodiments, such antisense activity is a change in the ratio of nucleic acid or protein splice variants. In certain embodiments, such antisense activity is a phenotypic change in the cell and / or subject.

  The “detection” or “measurement” of the antisense activity may be direct or indirect. For example, in certain embodiments, antisense activity is assessed by detecting and / or measuring the amount of target nucleic acid or protein or the relative amount of a splice variant of the target nucleic acid or protein. In certain embodiments, antisense activity is detected by observing phenotypic changes in the cell or animal. In connection with any activity, reaction, or action, the terms “detection” and “measurement” indicate that a test for detection or a test for measurement is performed. Such detection and / or measurement may include a value of zero. Thus, even if the result of a test for detection or a test for measurement results in no activity being found (zero activity), the step of detecting or measuring the activity is nevertheless Done.

  “Target nucleic acid” refers to any nucleic acid molecule capable of modulating its expression, amount, or activity by an antisense compound.

  “Target mRNA” refers to a preselected RNA molecule that encodes a protein.

  “Target pre-mRNA” means a preselected RNA transcript that has not been completely processed into mRNA. In particular, pre-mRNA includes one or more introns.

  “Target protein” means a protein encoded by a target nucleic acid.

  “Modulation” means a change in function or activity. In certain embodiments, modulation means an increase in gene expression. In certain embodiments, modulation means a decrease in gene expression.

  “Expression” means any function and process by which information encoded in a gene is converted into a structure that exists and functions in a cell.

  “Nucleobase sequence” means the order of consecutive nucleobases in the 5 ′ to 3 ′ direction, regardless of any sugar, linkage and / or nucleobase modification.

  “Contiguous nucleobases” means nucleobases located immediately adjacent to each other in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases to add non-covalently via hydrogen bonds.

  “Complementary” means that a first nucleic acid can hybridize to a second nucleic acid under stringent hybridization conditions. For example, an antisense compound is complementary to its target nucleic acid if it can hybridize to the target nucleic acid under stringent hybridization conditions.

“Completely complementary” means that each nucleobase of a first nucleic acid can be appended with a nucleobase at each corresponding contiguous position of a second nucleic acid.

  By “% complementarity” of an antisense compound is meant the percentage of the nucleobase of the antisense compound that is complementary to an equal length portion of the target nucleic acid. % Complementarity is calculated by dividing the number of nucleobases of the antisense oligonucleotide complementary to the nucleobases at corresponding consecutive positions in the target nucleic acid by the total length of the antisense compound.

  “% Identity” means the number of nucleobases in the first nucleic acid that are identical to the nucleobase at the corresponding position in the second nucleic acid divided by the total number of nucleobases in the first nucleic acid To do.

  “Hybridize” refers to the annealing of complementary nucleic acids that produce complementarity through nucleobases.

A “mismatch” cannot pair with a nucleobase at the corresponding position in the second nucleic acid,
Means the nucleobase of the first nucleic acid.

  “Identical nucleobase sequence” means having the same nucleobase sequence regardless of any chemical modification of the nucleoside.

“Different modifications” or “differently modified” refers to nucleoside or internucleoside linkages having different nucleoside modifications or internucleoside linkages, such as not including modifications. Thus, for example, an MOE nucleoside and an unmodified DNA nucleoside are “modified differently” even though the DNA nucleoside is not modified. Similarly, DNA and RNA are “differently modified” even though both are naturally occurring unmodified nucleosides. Identical nucleosides that contain different nucleobases are not modified differently unless otherwise stated. For example, a nucleoside containing a 2′-OMe modified sugar, an adenine nucleobase, a nucleoside containing a 2′-OMe modified sugar, and a thymine nucleobase are not modified differently.

“Identical modification” means nucleoside and internucleoside linkages (including unmodified nucleoside and internucleoside linkages) that are identical to each other. Thus, for example, two unmodified DNA nucleosides are those having “identical modifications” even though the DNA nucleoside is unmodified.

A “modified type” or some “type” of nucleoside means a modification of the nucleoside, and includes modified and unmodified nucleosides. Thus, unless otherwise specified, a “nucleoside having a first type of modification” may be an unmodified nucleoside.

“Distinct region” of an oligonucleotide means a portion of an oligonucleotide in which all nucleoside and internucleoside linkages within a region all contain the same modification; and any nucleoside and / or internucleoside of any adjacent portion. Binding involves at least one different modification.

  “Motif” means a pattern of modified and / or unmodified nucleobases, sugars, and / or internucleoside linkages in an oligonucleotide.

  “Fully modified oligonucleotide” means that each nucleobase, each sugar, and / or each internucleoside linkage is modified.

  “Homogeneously modified oligonucleotide” means that each nucleobase, each sugar, and / or each internucleoside linkage has the same modification throughout the modified oligonucleotide.

"Alternating motif" is at least four different regions of the modified nucleoside refers to an oligonucleotide, or a portion thereof having a pattern (AB) _n A _m, where A is a nucleoside region having a first modified B; nucleoside regions with different modified types; n is 2-15; and m is 0 or 1. Thus, in certain embodiments, alternating motifs include 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or More alternating areas are included. In certain embodiments, each A
The region and each B region independently contains 1 to 4 nucleosides.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Subject in need thereof” means a subject identified as in need of treatment or treatment. In such embodiments, the subject has one or more indications that have or develop SMA.

  “Administering” means providing a pharmaceutical agent or composition to a subject, and this concept includes when administered by a medical professional and when administered by himself However, it is not limited to these.

  “Parenteral administration” means administration via injection or infusion.

  Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

  “Systemic administration” means administration to a region other than the intended site of activity. Examples of systemic administration are subcutaneous and intravenous administration, and intraperitoneal administration.

  “Subcutaneous administration” means administration directly under the skin.

  “Intravenous administration” means administration into a vein.

  “Cerebrospinal fluid” or “CSF” means a fluid that fills the spaces around the brain and spinal cord.

“Administration into the cerebrospinal fluid” means any administration that delivers the substance directly into the CSF.

  “Intraventricular” or “ICV” means administration to the ventricular system of the brain.

  “Subarachnoid space” or “IT” means administration into the CSF under the arachnoid membrane that covers the brain and spinal cord. IT injections are made through the outer membrane of the spinal cord and into the subarachnoid space, where a pharmaceutical agent is injected into the sheath surrounding the spinal cord.

“Induction phase” means the administration phase where administration is initiated and a steady state concentration of the active pharmaceutical agent is achieved in the target tissue. For example, the induction phase is the administration phase when a steady state concentration of antisense oligonucleotide is achieved in the liver.

  “Maintenance phase” means the administration phase after a steady state drug concentration of the target tissue has been obtained.

“Period” means a period of time during which activity or decline continues. For example, the period of the induction phase is the period during which the induction dose is administered.

  “Maintenance dose” means the dose administered in a single dose during the maintenance phase. As used herein, “induction dose” means the dose administered in a single dose during the induction phase.

  “Simultaneous administration” means that two or more pharmaceutical agents are administered to a subject. The two or more pharmaceutical agents may be in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents can be administered via the same or separate routes of administration. Co-administration includes simultaneous or sequential administration.

  “Treatment” means a method of treating a disease. In certain embodiments, treatment includes, but is not limited to, surgical treatment, clinical treatment, and physical interventions such as assisted breathing, feeding tubes, and physical therapy aimed at increasing strength. Not.

  “Treatment” means applying one or more specific procedures used to cure or ameliorate a disease. In certain embodiments, the specific procedure is to administer one or more pharmaceutical agents.

“Improvement” means reducing the severity of at least one indicator of a symptom or disease. In certain embodiments, the improvement includes delaying or delaying the progression of one or more indicators of symptoms or disease. The severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art.

  “Suppress the onset of” means to suppress the occurrence of a symptom or disease in a subject at risk of developing the disease or symptom. In certain embodiments, a subject at risk of developing a disease or condition is given a treatment similar to that given to a subject who already has the disease or condition.

  “Delaying the onset of” means delaying the onset of a symptom or disease in a subject at risk of developing the disease or symptom.

“Slowing the progression of” means that the severity of a disease or symptom of at least one symptom worsens more slowly.

“Exon 7 amino acid” means the portion of the SMN protein corresponding to exon 7 of SMN RNA. The amino acid of exon 7 is present in the SMN protein expressed from SMN RNA where exon 7 has not been removed during splicing.

  “SMN protein” means normal full-length survival motor neuron protein. SMN can be expressed from either the SMN1 gene or the SMN2 gene so long as exon 7 is present in the mature mRNA and the amino acid of exon 7 is present in the SMN protein .

“Dose” means a defined amount of a pharmaceutical agent provided in a single dose or over a defined time. In certain embodiments, the dose can be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous or subarachnoid or ICV administration is preferred, the desired dose requires a dose that cannot be readily provided by a single injection. In such embodiments, two or more injections can be used to achieve the desired dose. In a continuous infusion setting, the dose can be expressed as the amount of pharmaceutical agent delivered per unit time.

“Dosage unit” means a form in which a pharmaceutical agent is provided. In certain embodiments, the dosage unit is a vial containing lyophilized oligonucleotide. In certain embodiments, the dosage unit is a vial containing the reconstituted oligonucleotide.

  “Therapeutically effective amount” means the amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.

  “Pharmaceutical composition” means a mixture of substances suitable for administration to an individual, including a pharmaceutical agent. For example, the pharmaceutical composition may comprise a modified oligonucleotide and a sterile aqueous solution.

  “Acceptable safety profile” means a pattern of side effects within clinically acceptable limits.

  “Side effect” means a physiological response other than the desired effect that can contribute to the treatment.

1. Certain modified oligonucleotides In certain embodiments, the present invention relates to antisense oligonucleotides comprising one or more modifications compared to oligonucleotides of naturally occurring oligomers, such as DNA or RNA. Related methods and compositions are provided. Such modified antisense oligonucleotides can have one or more desired properties. Certain such modifications, for example, by increasing its resistance to one or more nucleases, by increasing the affinity of the antisense oligonucleotide to its target nucleic acid,
And / or altering the antisense activity of the antisense oligonucleotide by altering the pharmacokinetics or tissue distribution of the oligonucleotide. In certain embodiments, such modified antisense oligonucleotides comprise one or more modified nucleosides and / or one or more modified nucleoside linkages and / or one or more complex groups.

a. Certain Modified Nucleosides In certain embodiments, the antisense oligonucleotide comprises one or more modified nucleosides. Such modified nucleosides may include modified sugars and / or modified nucleobases. In certain embodiments, incorporation of such modified nucleosides into the oligonucleotide results in increased affinity for the target nucleic acid and / or increased resistance to nuclease degradation, and / or improved toxicity of the modified oligonucleotide and Results in increased stability, including but not limited to improved uptake properties.

i. Certain Nucleobases The base portion of naturally occurring nucleosides is a heterocyclic base, typically purines and pyrimidines. Purine nucleobases adenine (A) and guanine (G), and pyrimidine nucleobases thymi (T), cytosine (C) and uracil (U), etc. to “unmodified” or “natural” nucleobases In addition, a number of modified nucleobases or nucleobase mimetics known to those skilled in the art are suitable for incorporation into the preferred compounds herein. In certain embodiments, the modified nucleobase is a nucleobase that is substantially similar in structure to the parent nucleobase such as, for example, 7-deazapurine, 5-methylcytosine, or G-clamp. In certain embodiments, nucleobase mimetics include more complex structures such as, for example, tricyclic phenoxazine nucleobase mimetics. Methods for preparing the above-described modified nucleobases are also known to those skilled in the art.

ii. Certain modified sugars and sugar substitutes The antisense oligonucleotides of the present invention may optionally contain one or more nucleosides, wherein the sugar component is modified compared to the natural sugar. Has been. Oligonucleotides containing such sugar-modified nucleosides may have increased nuclease stability, increased binding affinity, or increased some other beneficial biological property. Such modifications include the addition of substituents, bridges of ring atoms that are not geminals (two atoms of the same type attached to one atom) to form a bicyclic nucleic acid (BNA), S, N ( R), or substitution of a ribosyl ring oxygen atom with C (R ₁ ) (R) ₂ (R = H, C ₁ -C ₁₂ alkyl or protecting group), and for example, 2′-F-5′-methyl substituted nucleosides Combinations of these (see PCT International Application 2008/101157, for other disclosed 5 ′, 2′-bis substituted nucleosides published on August 21, 2008), or even 2′-position substituents Substitution of a ribosyl ring oxygen atom with S (see published US patent application US2005-0130923, published Jun. 16, 2005), or selective 5′-substitution of BNA (PCT international application WO 2007/134181) And published on Nov. 22, 2007, LNA is substituted, for example with a 5'-methyl or 5'-vinyl group). But it is not limited to, et al.

Examples of nucleosides with modified sugar moieties include 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ₃ and 2'-O (CH ₂ ) Nucleosides containing ₂ OCH ₃ substituents are included, but are not limited to. Substituents at the 2 ′ position are allyl, amino, azide, thio, O-allyl, OC ₁ -C ₁₀ alkyl, OCF ₃ , O (CH ₂ ) ₂ SCH ₃ , O (CH ₂ ) ₂ -ON (R _m ) (R _n ), and O—CH ₂ —C (═O) —N (R _m ) (R _n ), where each R _m and R _n are independently , H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

Examples of bicyclic nucleic acids (BNA) include, but are not limited to, nucleosides that contain a bridge between the 4 'and 2' ribosyl ring atoms. In certain embodiments, the antisense compounds provided herein include one or more BNA nucleosides, wherein the bridge is of the formula: 4′-β-D- (CH ₂ ) -O-2 '(β-D-LNA);4'-(CH ₂ ) -S-2 ';4'-α-L- (CH ₂ ) -O-2'(α-L-LNA); _{4 '- (CH 2) 2} -O-2'(ENA);4'-C (CH 3) 2 -O-2 '( see PCT / US2008 / 068922); 4' -CH (CH 3) - O-2 ′ and 4′-CH (CH ₂ OCH ₃ ) —O-2 ′ (see US Pat. No. 7,399,845 issued July 15, 2008); 4′-CH ₂ —N (OCH ₃ ) -2 '(see PCT / US2008 / 064591); 4'-CH ₂ -ON (CH ₃ ) -2' (published US patent application US2004-0171570 issued September 2, 2004)
4′-CH ₂ —N (R) -O-2 ′ (US Pat. No. 7,427,672 issued on September 23, 2008); 4′-CH ₂ —C (CH ₃ ) -2 ′ and _{4'-CH 2 -C - (=} CH 2) -2 ' includes one of (see PCT / US2008 / 066154); and R is independently, H, C ₁ -C ₁₂ alkyl, Or a protecting group.

In certain embodiments, the present invention includes modified nucleosides that include modified sugar components that are not bicyclic sugar components. Certain such modified nucleosides are known. In certain embodiments, the sugar ring of the nucleoside may be modified with any one. Examples of sugar modifications useful in the present invention include sugar substituents selected from the following: OH, F, O-alkyl, S-alkyl, N-alkyl, or O-alkyl-O-alkyl. compounds are included, wherein the alkyl, the alkenyl and alkynyl are substituted C ₁ may be unsubstituted even if -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl, however are limited to Do not mean. In certain such embodiments, such substituents are those at the 2 ′ position of the sugar.

In certain embodiments, the modified nucleoside includes a substituent at the 2 ′ position of the sugar. In certain embodiments, such substituents include the following: halides (including but not limited to F), allyl, amino, azide, thio, O-allyl, OC ₁ -C ₁₀ alkyl, — _{OCF 3, O- (CH 2)} 2 -O-CH 3, 2'-O (CH 2) 2 SCH 3, O- (CH 2) 2 -ON (R m) (R n), or O-CH2 -C (= O) -N (R m) (R n), are selected from, wherein each of R _m and R _n, are independently, C ₁ or is not being replaced are H or substituted - C ₁₀ alkyl.

In certain embodiments, suitable modified nucleosides for use in the present invention are: 2-methoxyethoxy, 2′-O-methyl (2′-O—CH ₃ ), 2′-fluoro (2′-F) is there.

In certain embodiments, the modified nucleoside is the following at the 2′-position: O [(CH ₂ ) _n O] _m CH ₃
, O (CH ₂ ) _n NH ₂ , O (CH ₂ ) _n CH ₃ , O (CH ₂ ) _n ONH ₂ , OCH ₂ C (═O) N (H) CH ₃ , and O (CH ₂
) _N ON [(CH ₂ ) _n CH ₃ ] ₂ , wherein n and m are 1 to about 10. Other 2'-sugar substituent, the following ones: C ₁ -C ₁₀ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O- aralkyl, SH, SCH _3, OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted Includes silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving pharmacokinetic properties, or groups for improving the pharmacodynamic properties of oligomeric compounds, and other substituents with similar properties .

In certain embodiments, the modified nucleoside comprises a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitutions have improved binding affinity compared to unmodified nucleosides and other modified nucleosides such as 2'-O-methyl, O-propyl, and O-aminopropyl. As described. Oligonucleotides with 2'-MOE substituents have also been shown to be antisense inhibitors of gene expression with a promising future for in vivo applications (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.
Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides or Nucleotides, 1997, 16, 917-926).

In certain embodiments, the 2′-sugar substituent is either in the arabino (up) position or the ribo (down) position. In certain such embodiments, the 2'-arabino modification is 2'-F arabino (FANA
). Similar modifications can be made at other positions on the sugar, particularly the 3 'position of the sugar of the 3' terminal nucleoside, or the 5 'position of the 2'-5' linked oligonucleotide and the 5 'terminal nucleotide.

In certain embodiments, nucleosides suitable for use in the present invention have sugar substitutes such as cyclobutyl instead of ribofuranosyl sugars. Representative US patents that teach the preparation of such modified sugar structures include: US: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is hereby incorporated by reference in its entirety, but is not limited thereto is not.

In certain embodiments, the present invention provides a nucleoside comprising a modification at the 2′-position of the sugar. In certain embodiments, the present invention provides a nucleoside comprising a modification at the 5′-position of the sugar. In certain embodiments, the invention provides nucleosides that include modifications at the 2′- and 5′-positions of the sugar. In certain embodiments, modified nucleosides may be useful for incorporation into oligonucleotides. In certain embodiments, the modified nucleoside incorporates an oligonucleoside at the 5′-end of the oligonucleotide.

b. Specific Internucleoside Bonds Antisense oligonucleotides of the invention may optionally contain one or more modified internucleoside bonds. These two major classes of linking groups are defined by the presence or absence of a phosphorus atom. Exemplary phosphorus-containing linkages include, but are not limited to, phosphodiester (P = O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P = S). . Typical non-phosphorus containing linking groups include methylenemethylamino (—CH ₂ —N (CH ₃ ) —O—CH ₂ —), thiodiester (—OC (O) —S—), thionocarbamate ( -OC (O) (NH) -S- ); siloxane (-O-Si (H) 2 -O-); and N, N'-dimethylhydrazine _{(-CH 2 -N (CH 3)} -N (CH ₃ )-) includes, but is not limited to. Oligonucleotides with non-phosphorous linkage groups are called oligonucleosides. Modified linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphodiester linkages. In certain embodiments, a bond with a chiral atom can be prepared as a racemic mixture, as a separate enantomer. Exemplary chiral linkages include, but are not limited to alkyl phosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known to those skilled in the art.

The antisense oligonucleotides described herein contain one or more asymmetric centers so that (R) or (S) such as a sugar anomer, or (D) or ( L) results in enantiomeric, diastereomeric, and other stereoisomeric structures that can be defined in terms of absolute stereochemistry. The antisense compounds provided herein include all such possible isomers, as well as their racemates and optionally its pure forms.

In certain embodiments, the antisense oligonucleotide has at least one modified internucleoside linkage. In certain embodiments, the antisense oligonucleotide has at least two modified internucleoside linkages. In certain embodiments, the antisense oligonucleotide has at least 3 modified internucleoside linkages. In certain embodiments, the antisense oligonucleotide has at least 10 modified internucleoside linkages. In certain embodiments, each internucleoside linkage of an antisense oligonucleotide is a modified internucleoside linkage. In certain embodiments, such modified internucleoside linkage is a phosphorothioate linkage.

c. Length In certain embodiments, the present invention provides antisense oligonucleotides of any of a range of lengths. In certain embodiments, the present invention provides an antisense compound or antisense oligonucleotide comprising or consisting of an XY linked nucleoside, wherein X and Y are each independently 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, Selected from 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X <Y. For example, in certain embodiments, the present invention is: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18 , 8-19, 8-20, 8-21, 8-22, 8-23
, 8-24, 8-25, 8-26, 8-27, 8-28, 8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9 -15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27 9-28, 9-29, 9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10 -20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27, 10-28, 10-29, 10-30, 11-12, 11-13 11-14 14, 11-15, 11-16, 11-17, 11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11 -26, 11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17, 12-18, 12
-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26, 12-27, 12-28, 12-29, 12-30, 13-14 , 13-15, 13-16, 13-17, 13-18, 13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13 -27, 13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20, 14-21, 14-22, 14-23 , 14-24, 14-25, 14-26, 14-27, 14-28, 14-29, 14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15 -21, 15-22, 15-23, 15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18, 16-19 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27, 16-28, 16-29, 16-30, 17-18, 17 -19, 17-20, 17-21, 17-22, 17-23, 17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19 , 18-20, 18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 19-20, 19 -21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29, 19-28, 19-29, 19-30, 20-21, 20-22, 20-23 , 20-24, 20-25, 20-26, 20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26, 21 -27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27, 22-28, 22-29, 22-30, 23-24 , 23-25, 23-26, 23-27, 23-28, 23-29, 23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25 -26, 25-27, 25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29, 27-30, 28-29 , 28-30, or 29-30 linked nucleosides or antisense compounds or antisense oligonucleotides comprising such nucleosides are provided.

  In certain embodiments, the antisense compounds or antisense oligonucleotides of the invention are 15 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the invention are 16 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the invention are 17 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the invention are 18 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the invention are 19 nucleosides in length. In certain embodiments, the antisense compounds or antisense oligonucleotides of the invention are 20 nucleosides in length.

d. Certain Oligonucleotide Motifs In certain embodiments, antisense oligonucleotides have chemically modified subunits that are arranged in specific directions along their length. In certain embodiments, the antisense oligonucleotides of the invention are fully modified. In certain embodiments, the antisense oligonucleotides of the invention are homogeneously modified. In certain embodiments, the antisense oligonucleotides of the invention are homogeneously modified and each nucleoside comprises a 2'-MOE sugar moiety. In certain embodiments, the antisense oligonucleotides of the invention are homogeneously modified and each nucleoside comprises a 2'-OMe sugar moiety. In certain embodiments, the antisense oligonucleotides of the invention are homogeneously modified and each nucleoside comprises a morpholino sugar moiety.

In certain embodiments, the oligonucleotides of the invention comprise alternating motifs. In certain such embodiments, the alternating modified form is selected from 2′-MOE, 2′-F, a bicyclic sugar-modified nucleoside, and DNA (unmodified 2′-deoxy). In certain such embodiments, each alternating region comprises a single nucleoside.

  In certain embodiments, the oligonucleotides of the invention comprise one or more blocks of a first type of nucleoside and one or more blocks of a second type of nucleoside.

In certain embodiments, the one or more alternating regions in the alternating motif include more than one type of single nucleoside. For example, the oligomeric compounds of the present invention may include one or more regions of any of the following nucleoside motifs:
Nu ₁ Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₁ ;
Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₂ Nu ₂ ;
Nu ₁ Nu ₁ Nu ₂ Nu ₁ Nu ₁ Nu ₂ ;
Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₁ Nu ₂ Nu ₂ ;
Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₁ ;
Nu ₁ Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₂ ;
Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₁ ;
Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₁ ;
Nu ₂ Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₁ ; or
Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₁ Nu ₂ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₂ Nu ₁ Nu ₁ ;
Here, Nu ₁ is a first type of nucleoside and Nu ₂ is a second type of nucleoside. In certain embodiments, one of Nu ₁ and Nu ₂ is a 2′-MOE nucleoside and the other of Nu ₁ and Nu ₂ is: 2′-OMe modified nucleoside, BNA, and unmodified DNA or RNA
Selected from nucleosides.

2. Oligomer Compound In certain embodiments, the present invention provides an oligomeric compound. In certain embodiments, the oligomeric compound consists solely of oligonucleotides. In certain embodiments, the oligomeric compound comprises an oligonucleotide and one or more conjugates and / or end groups. Such conjugates and / or end groups can be added to oligonucleotides having any of the chemical motifs described above. Thus, for example, an oligomeric compound comprising an oligonucleotide having alternating nucleoside regions may comprise a terminal group.

a. Specific Complex Groups In certain embodiments, the oligonucleotides of the invention are modified by the attachment of one or more complex groups. In general, complex groups include, but are not limited to, pharmacodynamics, pharmacokinetics, stability, binding, adsorption, cell distribution, cell uptake, charge, and clearance of oligomeric compounds that bind. Modifies one or more characteristics. Complex groups are commonly used in the chemical field and are attached to a parent compound, such as an oligomeric compound such as an oligonucleotide, via a direct or selective complex binding component or complex binding group. To do. Complex groups include intercalator, reporter molecule, polyamine, polyamide, polyethylene glycol, thioether, polyether, cholesterol, thiocholesterol, cholic acid component, folic acid, lipid, phospholipid, biotin, phenazine, phenanthridine, anthraquinone, These include, but are not limited to, adamantane, acridine, fluorescein, rhodamine, coumarin, and dye. Specific complex groups are those previously described, for example: cholesterol component (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al. al.,
Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain, For example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al ., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995) , 14, 9 69-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl component (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), Or octadecylamine or hexylamino-carbonyl-oxycholesterol component (Crooke et al.,
J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

In certain embodiments, the complex group is an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbutane, ketoprofen, (S)-(+)-planoprofen, capprofen, dansylsarcosine, 2 , 3,5-triiodobenzoic acid, flufenamic acid, folic acid, benzothiadiazole, chlorothiazide, diazepine, indomethacin, barbiturate, cephalosporin, sulfa drugs, antidiabetics, antibacterials or antibiotics. Oligonucleotide-drug conjugates and their preparation are described in US
It is described in patent application 09 / 334,130.

Representative US patents describing the preparation of oligonucleotide conjugates include: US: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830
; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726 5,597,696; 5,599,923; 5,599,928; and 5,688,941 but are not limited to these.

  The complex group can be attached to either or both of the end of the oligonucleotide (terminal complex group) and / or any inner portion.

b. End groups In certain embodiments, the oligomeric compounds comprise end groups at one or both ends. In certain embodiments, the end group may comprise any of the complex groups described below. In certain embodiments, the terminal group may comprise additional nucleosides and / or reverse abasic nucleosides. In certain embodiments, the end group is a stabilizing group.

In certain embodiments, the oligomeric compound comprises one or more terminal stabilizing groups that enhance properties such as, for example, nuclease resistance. Stabilizing groups include cap structures. The term “cap structure” or “end cap component”, as used herein, refers to a chemical modification that attaches to one or both ends of an oligomeric compound. Certain such end modifications can protect oligomeric compounds having terminal nucleic acid components from exonucleolytic degradation and assist in delivery and / or localization into the cell. The cap may be at the 5′-end (5′-cap), 3′-end (3′-cap), or at both ends. (For further details, see Wincott et al., International PCT Publication No. WO 97/26270; Beaucage and Tyer, 1993, Tetrahedron.
49, 1925; US Patent Application Publication No. US 2005/0020525; and WO 03/004602).

In certain embodiments, one or more additional nucleosides are added to one or both ends of the oligonucleotide of the oligomeric compound, and such additional terminal nucleosides are referred to herein as terminal group nucleosides. In double-stranded compounds, such end group nucleosides are terminal (3 ′ and / or 5 ′) overhangs. In the setting of double-stranded antisense compounds, such end group nucleosides may or may not be complementary to the target nucleic acid. In certain embodiments, the end group is a non-nucleoside end group. Such a non-terminal group may be any terminal group other than a nucleoside.

c. Oligomer compounds In a particular embodiment, the oligomeric compounds of the invention comprise a motif:
T _1- (Nu ₁ ) _n1- (Nu ₂ ) _n2- (Nu ₁ ) _n3- (Nu ₂ ) _n4- (Nu ₁ ) _n5 -T ₂ ,
Including, where:
Nu ₁ is the first type of nucleoside;
Nu ₂ is the first type of nucleoside;
each of n1 and n5 is independently 0-3;
the sum of n2 + n4 ranges from 10 to 25;
n3 is 0-5; and
Each of T ₁ and T ₂ is independently H, a hydroxyl protecting group, a selective binding complex group or a cap group.

  In certain such embodiments, the sum of n2 + n4 is 13 or 14, n1 is 2, n3 is 2 or 3, and n5 is 2. In certain such embodiments, the oligomeric compounds of the invention comprise a motif selected from Table A.

Table A is intended to illustrate the invention, but does not limit the invention. The oligomeric compounds shown in Table A each contain 20 nucleosides. Oligomeric compounds containing more or fewer nucleosides can be readily prepared by selecting different numbers of nucleosides for one or more of n1-n5. In certain embodiments, Nu ₁ and Nu ₂ are each selected from: 2′-MOE, 2′-OMe, DNA, and bicyclic nucleosides.

3. Antisense In a particular embodiment, the oligomeric compound of the invention is an antisense compound. Thus, in such embodiments, the oligomeric compound hybridizes with the target nucleic acid, resulting in antisense activity.

a. Hybridization In certain embodiments, the invention provides for conditions under which specific binding is desired, i.e., physiological conditions for in vivo assays or therapeutic treatments, and when the assay is an in vitro assay. An antisense compound that specifically hybridizes to the target nucleic acid when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to the non-target nucleic acid sequence under the conditions under which it occurs I will provide a.

Thus, “stringent hybridization conditions” or “stringent conditions” means conditions under which an antisense compound hybridizes to a target sequence but hybridizes to a minimal number of other sequences. Stringent conditions are sequence-dependent and are different under different conditions, and “stringent conditions” under which an antisense oligonucleotide hybridizes to a target sequence are those of the antisense oligonucleotide. Determined by assays that study the properties and composition and antisense oligonucleotides.

Nucleotide incorporation, affinity, and modification similarly tolerate more mismatches compared to unmodified compounds, and certain nucleobase sequences are more tolerant of mismatches than other nucleobase sequences. It is understood in the art that this is possible. One skilled in the art can determine an appropriate number of mismatches between oligonucleotides or between an antisense oligonucleotide and a target nucleic acid, eg, by determining the melting temperature (Tm). Tm or ΔTm can be calculated by techniques well known to those skilled in the art. For example, by the technique described in Freier et al. (Nucleic Acids Research, 1997, 25, 22: 4429-4443), one skilled in the art can determine the ability of nucleotide modifications to increase the melting temperature of RNA: DNA duplexes. Nucleotide modifications can be evaluated.

b. Pre-mRNA Processing In certain embodiments, the antisense compounds provided herein are complementary to pre-mRNA. In certain embodiments, such antisense compounds are altered in pre-mRNA splicing. In certain such embodiments, the ratio of one variant of mature mRNA corresponding to the target pre-mRNA to another variant of mature mRNA is varied. In certain such embodiments, the ratio of one variant of the protein expressed from the target pre-mRNA to the other variant of the protein is varied. Specific oligomeric compounds and nucleobase sequences that can be used to alter pre-mRNA splicing are described, for example, in the following references (US 6,210,892; US 5,627,274; US 5,665,593; 5,916,808; US 5,976,879; US2006 / 0172962; US2007) US2005 / 0074801; US2007 / 0105807; US2005 / 0054836; WO 2007/090073; WO2007 / 047913; Hua et al., PLoS Biol 5 (4): e73; Vickers et al.,
J. Immunol. 2006 Mar 15, 176 (6): 3652-61; and Hua et al., American J. of Human
Genetics (April 2008) 82, 1-15), each of which is incorporated by reference in its entirety for all purposes. In certain embodiments, antisense sequences that alter splicing were modified according to the motifs of the invention.

Antisense is an effective means for modifying the expression of one or more specific gene products and is particularly useful in a number of therapeutic, diagnostic and research applications. Provided herein are antisense compounds useful for modifying gene expression via antisense mechanisms of action, including antisense mechanisms based on target occupancy. In one aspect, the antisense compounds provided herein modify target gene splicing. Such modulation includes promoting or inhibiting exon inclusions. Further provided herein are antisense compounds that target cis splicing control elements present in pre-mRNA molecules, including exon splicing enhancers, exon splicing silencers, intron splicing enhancers and intron splicing silencers. Disruption of the cis splicing control element is thought to change splicing site selection, which can result in a change in the composition of the splice product.

Eukaryotic pre-mRNA processing is a complex process that requires a large number of signal and protein factors to achieve proper mRNA splicing. Spliceosome exon definition requires more than the standard splicing signal that defines the intron-exon boundary. One such additional signal is provided by cis-acting regulatory enhancer and silencer sequences. Exon splicing enhancer (ESE), exon splicing silencer (ESS), intronic splicing enhancer (ISE) and intron splicing silencer (ISS) suppresses or enhances the use of splicing donor sites or splicing acceptor sites. Depending on the mode of action, it was identified (Yeo et al. 2004, Proc. Natl. Acad. Sci. USA 101 (44): 15700-15705). The binding of specific proteins (trans factors) to these regulatory sequences is either for the splicing process or either facilitates or represses the use of specific splicing sites and modifies the ratio of splicing products ( Scamborova et al. 2004, Mol. Cell. Biol. 24 (5): 1855-1869; Hovhannisyan and Carstens, 2005, Mol. Cell. Biol. 25 (1): 250-263; Minovitsky et al. 2005, Nucleic Acids Res. 33 (2): 714-724).

4. Pharmaceutical Composition In certain embodiments, the present invention provides a pharmaceutical composition comprising one or more antisense compounds. In certain embodiments, such pharmaceutical compositions comprise a sterile saline solution and one or more antisense compounds. In certain embodiments, such pharmaceutical compositions consist of a sterile saline solution and one or more antisense compounds.

  In certain embodiments, the antisense compound may be mixed with pharmaceutically acceptable active and / or inactive substances for the preparation of a pharmaceutical composition or formulation. Compositions and methods for formulating pharmaceutical compositions depend on a number of criteria including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

In certain embodiments, antisense compounds can be used in pharmaceutical compositions by combining such oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in parenteral delivery compositions. Accordingly, in certain embodiments, a pharmaceutical composition comprising an antisense compound and a pharmaceutically acceptable diluent is used in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS.

  Pharmaceutical compositions containing an antisense compound include any pharmaceutically acceptable salt, ester, or salt of such an ester. In certain embodiments, a pharmaceutical composition comprising an antisense compound provides a biologically active metabolite or residue thereof (directly or indirectly) when administered to an animal, including a human. It can contain one or more oligonucleotides. Thus, for example, the disclosure also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of antisense compounds. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

Prodrugs may include those incorporating an additional nucleoside at one or both ends of the oligomeric compound that is cleaved by endogenous nucleases in the body to form an active antisense oligomeric compound.

Lipid-based vectors have been used in nucleic acid therapy in various ways. For example, in one method, the nucleic acid is incorporated into a preformed liposome or a lipoplex consisting of a mixture of cationic and neutral lipids. In another method, a DNA complex comprising a mono-cationic lipid or a poly-cationic lipid is formed in the absence of neutral lipids.

Certain preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (01 May 2008
), Which is incorporated herein by reference in its entirety.

5. Administration to a subject In certain embodiments, a pharmaceutical composition comprising one or more antisense compounds is administered to a subject. In certain embodiments, such pharmaceutical compositions are administered by injection. In certain embodiments, such pharmaceutical compositions are administered by infusion.

In certain embodiments, the pharmaceutical composition is administered into the CSF by injection or infusion. In certain such embodiments, the pharmaceutical composition is administered by direct injection or infusion into the spinal cord. In certain embodiments, the pharmaceutical composition is administered by injection or infusion into the brain. In certain embodiments, the pharmaceutical composition is administered by subarachnoid infusion or infusion rather than to the spinal cord tissue itself. Without being limited by theory, in certain embodiments, the antisense compound can be released into the surrounding CSF and penetrated into the spinal cord parenchyma. An additional advantage of subarachnoid space delivery is that the subarachnoid route is similar to lumbar puncture administration that is already commonly used in humans (ie, spinal tap).

In certain embodiments, the pharmaceutical composition is administered by intracerebroventricular (ICV) injection or infusion. Intracerebroventricular administration, or intraventricular delivery, of pharmaceutical compositions comprising one or more antisense compounds can be performed in one or more ventricles, which are filled with cerebrospinal fluid (CSF) Is done. CSF is a clear fluid that fills the ventricle, resides in the subarachnoid space, and surrounds the brain and spinal cord. CSF is created by the choroid plexus and through the flow or transport of tissue fluid by the brain into the ventricles. The choroid plexus is a structure that lines the bottom of the lateral ventricle and the ceiling of the third and fourth ventricles. Particular studies have shown that these structures can produce 400-600 ccs of fluid per day, consistent with the amount to fill the central nerve space four times a day. In adults, the volume of this liquid is calculated to be 125-150 ml (4-5 oz). CSF is continuously produced, circulated and absorbed. Certain studies have shown that approximately 430-450 ml (approximately 2 cups) of CSF can be produced daily. From specific calculations, production is estimated to arrive at approximately 0.35 ml / min for adults and 0.15 ml / min for human infants. The lateral ventricular choroid plexus produces most of the CSF. CSF flows through the Monroe hole into the third ventricle, where product from the third ventricle is added and continues to descend into the fourth ventricle through the Sylvian aqueduct. The fourth ventricle adds more CSF; fluid then moves through the Majandhi and Rushka holes into the subarachnoid space. The fluid then circulates throughout the base of the brain, descends to the periphery of the spinal cord, and travels above both cerebral hemispheres. CSF enters the blood via the arachnoid villi and the intracranial vascular sinus.

  In certain embodiments, such pharmaceutical compositions are administered systemically. In certain embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments, the pharmaceutical composition is administered by intramuscular injection.

In certain embodiments, the pharmaceutical composition is administered directly to the CSF (eg, IT and / or ICV injection and / or infusion) and systemically.

In certain embodiments, systemically administered antisense compounds enter neurons. In certain embodiments, systemically administered antisense compounds are used, particularly in young subjects (eg, in utero subjects and / or neonatal subjects) where the blood-brain barrier is not completely formed. It can penetrate the blood-brain barrier. In certain embodiments, some amount of a systemically administered antisense compound can be taken up by neurons, even in a subject in which the blood-brain barrier is fully formed. For example, antisense compounds can be taken up by neurons at or near neuromuscular junctions (retrograde uptake). In certain embodiments, such retrograde uptake results in antisense activity within neurons, including but not limited to motor neurons, and the antisense activity within neurons provides a therapeutic effect.

In certain embodiments, systemic administration provides a therapeutic effect through antisense activity that occurs in cells and / or tissues other than neurons. Evidence suggests that functional SMN inside neurons is required for normal neuronal function, while the impact of reduced functional SMN in other cells and tissues has not been well characterized. In certain embodiments, antisense activity in non-neuronal cells results in restoration of SMN function in non-neuronal cells, which subsequently produces a therapeutic effect.

In certain embodiments, improved SMN function in non-neuronal cells provides improved neuronal cell function regardless of whether SMN function within the neuron is improved. For example, in certain embodiments, systemic administration of a pharmaceutical composition of the invention results in antisense activity in muscle cells. Such antisense activity in muscle cells can provide benefits for motor-neurons or neurons in general associated with muscle cells.
In such embodiments, muscle cells with restored SMN function can provide factors that improve neuronal survival and / or function. In certain embodiments, such antisense activity is independent of the benefit from antisense activity arising from antisense compounds within neurons. In certain embodiments, systemic administration of a pharmaceutical composition of the invention produces antisense activity in other non-neuronal cells, including cells that are not immediately associated with neurons. Such antisense activity in non-neuronal cells can improve neuronal function. For example, antisense activity in non-neuronal cells (eg, liver cells) can give rise to factors that improve neuronal function in that cell. Note: Since the term “antisense activity” includes both direct and indirect activity, the benefit to neuronal function is “antisense activity” even if the antisense compound does not enter the neuron.

In certain embodiments, systemic administration of the pharmaceutical composition produces a therapeutic effect that is independent of direct or indirect antisense activity within the neuron. Typically, in SMA settings,
Neuronal function is reduced, resulting in significant symptoms. Additional symptoms may arise from decreased SMN activity in other cells. Certain such symptoms may be masked by the relative severity of symptoms resulting from reduced neuronal function. In certain embodiments, systemic administration results in a restored or improved SMN function in non-neuronal cells. In certain such embodiments, such restored SMN function or improved SMN function in non-neuronal cells has a therapeutic effect. For example, in certain cases, subjects with SMA have diminished growth. Such a decrease in growth may not result in a decrease in neuronal cell function. Indeed, reduced growth can cause a decrease in cellular function in another organ, such as the pituitary gland, and / or can result in SMN deficiency throughout the body cell. In such embodiments, systemic administration can result in improved SMN activity in pituitary cells and / or other cells, resulting in improved growth. In certain instances, administration to CSF restores sufficient neuronal function and allows the subject to live longer, however, since the subject lives longer, the subject is tested before such symptoms occur. One or more symptoms not previously known arise because the body generally dies. Certain such manifestations can be fatal. In certain embodiments, the manifesting symptoms are treated by systemic administration. Regardless of the mechanism, in certain embodiments, a variety of SMA symptoms, including but not limited to those previously masked by more severe symptoms associated with reduced neuronal function, are administered systemically. Can be treated.

In certain embodiments, systemic administration of the pharmaceutical composition of the invention results in increased SMN activity in muscle cells. In certain embodiments, such improvement in SMN activity in muscle cells is
Provides a therapeutic effect. Improvements in SMN activity only in muscle have been reported to be insufficient to provide a therapeutic effect (eg Gravrilina, et al., Hum Mol Genet 2008 17 (8): 1063-1075) . In certain embodiments, the present invention results in a method for improving intramuscular SMN function and in fact provides a therapeutic effect. In certain cases, the therapeutic effect can be attributed to improved SMN function in other cells (alone or in combination with muscle cells). In certain embodiments, an improvement in intramuscular SMN function alone can provide benefits.

  In certain embodiments, systemic administration results in improved survival.

6. Spinal muscular atrophy (SMA)
SMA is a genetic disorder characterized by the destruction of spinal motor neurons. SMA results from homozygous loss of both functional copies of the SMN1 gene. However, the SMN2 gene has the potential to encode the same protein as SMN1, and therefore SMA
Overcome the patient's gene deletion. SMN2 contains a translationally silent mutation (C → T) at position +6 of exon 7, which results in insufficient inclusion of exon 7 in the SMN2 transcript. Thus, the dominant form of SMN2, which lacks exon 7, is unstable and inactive. Thus, therapeutic compounds can modify SMN2 splicing to increase the proportion of SMN2 transcripts containing exon 7, and are useful for the treatment of SMA.

In certain embodiments, the present invention provides antisense compounds that are complementary to a pre-mRNA encoding SMN2. In certain such embodiments, the antisense compound alters SMN2 splicing. Specific sequences and regions useful for altering SMN2 splicing can be found in PCT / US06 / 024469, which is hereby incorporated by reference in its entirety for all purposes. In certain embodiments, an oligomeric compound having any of the motifs described herein has a nucleobase sequence that is complementary to intron 7 of SMN2. Certain such nucleobase sequences are exemplified in the following table, without limitation.

The antisense compounds of the present invention can be used to modify SMN2 expression in a subject such as a human. In certain embodiments, the subject has spinal muscular atrophy. In such specific subjects, the SMN1 gene is absent or a sufficient amount of functional SMN
Inability to produce protein. In certain embodiments, an antisense compound of the invention effectively modifies splicing of SMN2, resulting in an increase in exon 7 content in the SMN2 mRNA, and ultimately includes an amino acid corresponding to exon 7 SMN2 protein is produced. Such an alternative SMN2 protein is similar to the wild-type SMN protein. Antisense compounds of the present invention that effectively modify SMN2 mRNA expression or protein expression products are considered active antisense compounds.

Modulation of SMN2 expression can be measured in animal cells; animal tissues; or body fluids that may or may not contain animal organs. Body fluids (eg saliva, serum, CSF),
Methods for obtaining samples for analysis, such as tissues (eg, biopsies), or organs, and methods for preparing samples that allow analysis are well known to those skilled in the art. Methods for analysis of RNA and protein levels are as described above and are well known to those skilled in the art. The effect of the treatment is within the fluids, tissues or organs described above, recovered from animals contacted with one or more compounds of the present invention by common clinical methods known in the art. It can be evaluated by measuring biomarkers associated with target gene expression.

Also contemplated are methods of contacting a bodily fluid, organ or tissue with an effective amount of one or more antisense compounds or compositions of the invention. A bodily fluid, organ or tissue can be contacted with one or more compounds of the present invention resulting in modulation of SMN2 expression in the cells of the bodily fluid, organ or tissue. An effective amount can be determined by monitoring the modifying effect of the antisense compounds or compounds or compositions on the target nucleic acids or their products by methods common to those skilled in the art.

The present invention also provides an antisense compound as described herein for use in any of the methods described herein above. For example, the present invention is complementary to a nucleic acid encoding human SMN2 for use in treating a disease or condition associated with survival motor neuron protein (SMN), such as spinal muscular atrophy (SMA). An antisense compound is provided that includes a typical antisense oligonucleotide. As a further example, the present invention is used in treating a disease or condition associated with survival motor neuron protein (SMN) by administering an antisense compound directly into the central nervous system (CNS) or CSF. An antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid encoding human SMN2 is provided.

The invention also provides the use of an antisense compound as described herein in the manufacture of a medicament for use in any of the methods as described herein. For example, the present invention is complementary to a nucleic acid encoding human SMN2 in the manufacture of a medicament for treating a disease or condition associated with survival motor neuron protein (SMN), such as muscle atrophy (SMA). The use of antisense compounds, including antisense oligonucleotides, is provided. As a further example, the invention relates to the manufacture of a medicament for treating a disease or condition associated with survival motor neuron protein (SMN) by direct administration of the medicament into the central nervous system (CNS) or CSF. Provided is the use of an antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid encoding human SMN2.

  In certain embodiments, an oligomeric compound having any of the motifs described herein has a nucleobase sequence that is complementary to exon 7 of SMN2.

  In certain embodiments, an oligomeric compound having any of the motifs described herein has a nucleobase sequence that is complementary to intron 6 of SMN2.

In certain embodiments, the antisense compound comprises an antisense oligonucleotide having a nucleobase sequence comprising at least 10 nucleobases of the following sequence: TCACTTTCATAATGCTGG (SEQ ID NO: 1). In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 11 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 12 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 13 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 14 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 15 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 16 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising at least 17 nucleobases of such sequence. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence comprising such a sequence of nucleobases. In certain embodiments, the antisense oligonucleotide has a nucleobase sequence consisting of such sequences of nucleobases. In certain embodiments, an antisense oligonucleotide consists of 10-18 linked nucleosides and is a nucleobase sequence that is 100% identical to a portion of the same length of the following sequence: TCACTTTCATAATGCTGG (SEQ ID NO: 1) Have

7. Certain Subjects In certain embodiments, the subject has one or more indicators of SMA. In certain embodiments, the subject has reduced electrical activity of one or more muscles. In certain embodiments, the subject has a mutated SMN1 gene. In certain embodiments, the subject's SMN1 gene is absent or unable to produce a functional SMN protein. In certain embodiments, the subject is diagnosed by genetic testing. In certain embodiments, the subject is identified by muscle biopsy. In certain embodiments, the subject cannot sit straight. In certain embodiments, the subject cannot sit and / or walk. In certain embodiments, the subject needs assistance with breathing and / or diet. In certain embodiments, the subject is identified by electrophysiological measurement of muscle and / or muscle biopsy.

In certain embodiments, the subject has type I SMA. In certain embodiments, the subject has type II SMA. In certain embodiments, the subject has type III SMA. In certain embodiments, the subject is diagnosed as having SMA in the uterus. In certain embodiments, the subject is diagnosed as having SMA within one week of life. In certain embodiments, the subject is diagnosed as having SMA within one month of life. In certain embodiments, the subject is diagnosed as having SMA by 3 months of age. In certain embodiments, the subject is diagnosed as having SMA by 6 months of age. In certain embodiments, the subject is diagnosed as having SMA by 1 year of age. In certain embodiments, the subject is diagnosed with SMA in the range of 1-2 years. In certain embodiments,
Subjects are diagnosed with SMA in the range of 1-15 years. In certain embodiments, the subject is diagnosed as having SMA when the subject is 15 years of age or older.

In a particular embodiment, the first dose of the pharmaceutical composition according to the invention is administered intrauterine.
In certain such embodiments, the first dose is administered before the blood brain barrier is fully developed and shaved. In certain embodiments, the first dose is administered systemically and intrauterine to the subject. In certain embodiments, the first dose is administered intrauterine after formation of the blood brain barrier. In certain embodiments, the first dose is administered relative to CSF.

In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 1 week old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 1 month old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 3 months old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 6 months old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 1 year old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 2 years old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is less than 15 years old. In certain embodiments, the first dose of the pharmaceutical composition according to the invention is administered when the subject is 15 years of age or older.

8. Specific Dose In certain embodiments, the present invention provides dosages and frequency of administration. In certain embodiments, the pharmaceutical composition is administered as a bolus infusion. In certain such embodiments, the bolus infusion dose is 0.01 to 25 milligrams of antisense compound per kilogram body weight of the subject. In certain such embodiments, the bolus infusion dose is 0.01 to 10 milligrams of antisense compound per kilogram body weight of the subject. In certain embodiments, the dose is 0.05 to 5 milligrams of antisense compound per kilogram body weight of the subject. In certain embodiments, the dose is 0.1 to 2 milligrams of antisense compound per kilogram body weight of the subject. In certain embodiments, the dose is 0.5-1 milligram of antisense compound per kilogram body weight of the subject. In certain embodiments, such a dose is administered twice a month. In certain embodiments, such a dose is administered monthly. In certain embodiments, such doses are administered every 2 months. In certain embodiments, such a dose is administered every 6 months. In certain embodiments, such doses are administered by bolus injection into the CSF. In certain embodiments, such doses are administered by subarachnoid bolus injection. In certain embodiments, such doses are administered by bolus systemic injection (eg, subcutaneous injection, intramuscular injection, or intravenous injection). In certain embodiments, the subject receives a bolus infusion and bolus systemic injection into the CSF.
In such embodiments, the dose of CSF bolus and systemic bolus can be the same or different from each other. In certain embodiments, the CSF and systemic dose are administered at different frequencies. In certain embodiments, the present invention provides a dosage formulation comprising at least one bolus subarachnoid injection and at least one bolus subcutaneous injection.

In certain embodiments, the pharmaceutical composition is administered by continuous infusion. Such continuous infusion can be accomplished by an infusion pump that delivers the pharmaceutical composition to the CSF. In certain embodiments, such infusion pumps deliver pharmaceutical compositions by IT or ICV. In certain such embodiments, the dose administered ranges from 0.05 to 25 milligrams of antisense compound per kilogram body weight of the subject per day. In certain embodiments, the dose administered ranges from 0.1 to 10 milligrams of antisense compound per kilogram body weight of the subject per day. In certain embodiments, the dose administered is 0.5 to 10 milligrams of antisense compound per kilogram body weight of the subject per day. In certain embodiments, the dose administered is 0.5 to 5 milligrams of antisense compound per kilogram body weight of the subject per day. In certain embodiments, the dose administered is 1 to 5 milligrams of antisense compound per kilogram body weight of the subject per day. In certain embodiments, the present invention provides a dosage formulation comprising infusion into the CNS and at least one bolus systemic injection. In certain embodiments, the present invention provides a dosage formulation comprising instillation into the CNS and at least one bolus subcutaneous injection. In certain embodiments, doses are prepared to achieve or maintain a concentration of 0.1-100 micrograms of antisense compound per gram of CNS tissue, whether bolus or infusion. In certain embodiments, doses are prepared to achieve or maintain a concentration of 1-10 micrograms of antisense compound per gram of CNS tissue, whether bolus or infusion. In certain embodiments, doses are prepared to achieve or maintain a concentration of 0.1-1 microgram of antisense compound per gram of CNS tissue, whether bolus or infusion.

In certain embodiments, administration to a subject is divided into an induction phase and a maintenance phase. In certain such embodiments, the dose administered during the induction phase is greater than the dose administered during the maintenance phase. In certain embodiments, the dose administered during the induction phase is less than the dose administered during the maintenance phase. In certain embodiments, the induction phase is achieved by bolus injection and the maintenance phase is achieved by continuous infusion.

In certain embodiments, the present invention provides systemic administration of antisense compounds alone or in combination with delivery into CSF. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 200 mg / kg. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 100 mg / kg. In certain embodiments, the dose for systemic administration is 0.5 mg / kg to 100 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 100 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 50 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 25 mg / kg. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 25 mg / kg. In certain embodiments, the dose for systemic administration is 0.1 mg / kg to 10 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 10 mg / kg. In certain embodiments, the dose for systemic administration is 1 mg / kg to 5 mg / kg. In certain embodiments involving both systemic delivery and CSF delivery, the doses for the two routes are determined independently.

a. Calculation of Appropriate Human Dose In certain embodiments, the subject is a human. In certain embodiments, the human dose is calculated or estimated from data from animal experiments such as those described herein. In certain embodiments, the human dose is calculated or estimated from data from monkey and / or mouse experiments as described herein. In certain embodiments, the human dose is calculated or estimated from data from mouse experiments as described herein. In certain embodiments, an appropriate human dose can be calculated using pharmacokinetic data from mice in conjunction with knowledge of brain weight and / or cerebrospinal fluid (CSF) turnover rate. For example, a mouse's brain weight is approximately 0.4 g, which is approximately 2% of its body weight. In humans, the average brain weight is 1.5 kg, which is approximately 2.5% of its body weight. In certain embodiments, administration into CSF results in the elimination of some of the compound through uptake into brain tissue and subsequent metabolism. By using the ratio of human brain weight to mouse brain weight as a scale factor, removal through brain tissue and estimation of clearance can be calculated. In addition, the turnover rate of CSF can be used to estimate removal of the compound from the CSF into the blood. The turnover rate of mouse CSF is approximately 10-12 times per day (0.04 mL is produced at 0.325 μl / min). The human CSF turnover rate is approximately 4-fold per day (100-160 mL is produced at 350-400 μl / min). Clearance, and thus dosing requirements, can be based on brain weight removal scaling and / or CSF turnover scaling. Extrapolated human CSF clearance can be used to estimate human equivalent doses that approximate doses in mice. Thus, based on brain weight and CSF turnover rate, the human dose responsible for differences in tissue metabolism can be estimated. Such methods of calculation and estimation are known to those skilled in the art.

By way of non-limiting example, in certain embodiments, depending on the determined clearance and removal of a particular compound, the mg / kg mouse dose can be multiplied from the desired mouse dose by a factor of about 0.25 to about 1.25. Human equivalent doses can be estimated. Thus, for example, in certain embodiments, a human dose equivalent to a 0.01 mg dose for a 20 g mouse ranges from a total dose of about 8.75 mg to about 43.75 mg for a 70 kg human. Similarly, in certain embodiments, a human dose equal to a 0.01 mg dose for a 4 g newborn mouse ranges from a total dose of about 1.9 mg to about 9.4 mg for a 3 kg newborn human. These exemplary doses are merely illustrative of how one skilled in the art can determine appropriate human doses and are not intended to limit the invention.

In certain embodiments, human doses for systemic delivery (whether administered alone or in combination with CSF delivery) are derived from animal experiments such as those described herein. Calculated or estimated from the data. Typically, a suitable human dose (mg / kg) for a systemic dose is 0.1 to 10 times the effective volume of the animal. Thus, for example, a subcutaneous dose of 50 μg in a 2 g newborn mouse is a dose of 25 mg / kg. The corresponding dose for humans is expected to be 2.5 mg / kg to 250 mg / kg. For 3 kilograms of newborns, the corresponding dose is 7.5 mg to 750 mg. For a 25 kg child, the corresponding dose is 62.5 mg to 6250 mg.

9. Treatment Formulation In certain embodiments, the above-described dosage, administration frequency, route of administration, induction and maintenance phase, and timing of the first administration are combined to provide an administration formulation for a subject with SMA. Such dosage regimens are selected and adjusted to provide amelioration of one or more SMA symptoms and / or reduce or avoid toxicity or side effects that may contribute to administration of the pharmaceutical composition. In certain embodiments, the subject is in utero or is a newborn.
In such embodiments, administration of the pharmaceutical composition, particularly administration by continuous infusion, presents a particular challenge. Thus, in certain embodiments, the invention provides for administration of a pharmaceutical composition by bolus administration while the subject is in utero or very young, after which the subject is older and such If the installation of the pump is more practical, perform continuous infusion through an embedded infusion pump. Further, in certain embodiments, as the subject grows, the absolute dose is increased to achieve the same or similar dose: body weight ratio. The following table is intended to exemplify therapeutic formulations, but is not intended to limit the combinations that could be readily achieved by those skilled in the art.

   In certain embodiments, the dosage formulation includes systemic administration alone or in combination with administration into CSF (eg, Formula 3, above). The following table further illustrates such a formulation.

These therapeutic prescriptions are intended to be illustrative but not intended to limit the present invention. One skilled in the art can refer to the present disclosure and select an appropriate combination of dose and delivery based on various factors such as the severity of symptoms and general health and the age of the subject.

10. Co-administration In certain embodiments, the pharmaceutical composition of the invention is co-administered with at least one other pharmaceutical composition to treat SMA and / or to treat symptoms associated with one or more SMAs. Administer. In certain embodiments, such other pharmaceutical compositions are selected from trichostatin-A, valproic acid, riluzole, hydroxyurea, and butyrate or butyrate derivatives. In certain embodiments, the pharmaceutical composition of the invention is co-administered with trichostatin-A. In certain embodiments, the pharmaceutical composition of the invention is co-administered with a derivative of quinazoline, as described, for example, in Thurmond, et al., J. Med Chem. 2008, 51, 449-469. In certain embodiments, the pharmaceutical composition of the invention and at least one other pharmaceutical composition are co-administered simultaneously. In certain embodiments, the pharmaceutical composition of the present invention and at least one other pharmaceutical composition are co-administered at different times.

In certain embodiments, the pharmaceutical composition of the invention is co-administered with a gene therapy agent. In certain such embodiments, the gene therapy agent is administered to the CSF and the pharmaceutical composition of the invention is administered systemically. In certain such embodiments, the gene therapy agent is administered to CSF and the pharmaceutical composition of the invention is administered to CSF and systemically. In certain embodiments, the pharmaceutical composition of the invention and the gene therapy agent are co-administered simultaneously. In certain embodiments, the pharmaceutical compositions and gene therapy agents of the invention are co-administered at different times. Specific gene therapy approaches for SMA treatment have been reported (eg, Coady et al., PLoS ONE 2008 3 (10): e3468; Passini et al., J Clin Invest 2010 Apr 1, 120 (4): 1253- 64).

In certain embodiments, the pharmaceutical composition of the invention is co-administered with at least one other treatment for SMA. In certain embodiments, such other treatment for SMA is surgery. In certain embodiments, such other treatments are physical therapies that include (but are not limited to) exercises designed to strengthen muscles necessary for breathing, such as cough therapy. In certain embodiments, the other treatment is physical intervention, such as a feeding tube or a respiratory assist device.

  In certain embodiments, the pharmaceutical composition of the invention is co-administered with one or more other pharmaceutical compositions that reduce undesirable side effects of the pharmaceutical composition of the invention.

11. Phenotypic effects In certain embodiments, administration of at least one pharmaceutical composition of the invention results in a phenotypic change in the subject. In certain embodiments, such phenotypic changes include: Exon 7
Increased absolute amount of SMN mRNA containing exon; Increased ratio of SMN mRNA containing exon 7 to SMN mRNA lacking exon 7; Increased absolute amount of SMN protein containing exon 7; Exon 7 included Increased ratio of SMN protein to SMN protein lacking exon 7; improved muscle strength, improved electrical activity of at least one muscle; improved respiration; weight gain; and survival It is not done. In certain embodiments, at least one phenotypic change is detected in a motor neuron of the subject. In certain embodiments, administration of at least one pharmaceutical composition of the invention results in the subject being able to exercise, stand up and / or walk abdominal muscles. In certain embodiments, administration of at least one pharmaceutical composition of the invention results in the subject being able to eat, drink and / or breathe without assistance. In certain embodiments, the therapeutic effect is assessed by electrophysiological assessment of muscle. In certain embodiments, administration of a pharmaceutical composition of the invention ameliorates at least one symptom of SMA and has little or no inflammatory effect. In certain such embodiments, the absence of inflammatory effects is determined by the absence of a significant increase in Aif1 levels upon treatment.

In certain embodiments, administration of at least one pharmaceutical composition of the invention delays the onset of at least one symptom of SMA. In certain embodiments, administration of at least one pharmaceutical composition of the invention delays progression of at least one symptom of SMA. In certain embodiments, administration of at least one pharmaceutical composition of the present invention reduces the severity of at least one symptom of SMA.

In certain embodiments, administration of at least one pharmaceutical composition of the present invention results in undesirable side effects. In certain embodiments, the treatment regimen is identified to produce the desired reduction in symptoms while avoiding undesirable side effects.

12. Dosage Units In certain embodiments, the pharmaceutical compositions of the invention are prepared as dosage units for administration. A particular such dosage unit is one at a concentration selected from 0.01 mg to 100 mg. In certain such embodiments, the pharmaceutical composition of the invention is 0.01 mg, 0.1 mg, 0.5 mg, 1 mg
, 5 mg, 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, and 200 mg. In certain embodiments, the pharmaceutical composition comprises a dose of an oligonucleotide selected from 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 25 mg, and 50 mg.

13. Kits In certain embodiments, the present invention provides kits comprising at least one pharmaceutical composition. In certain embodiments, such kits further comprise a delivery means, such as a syringe or an infusion pump.

While the specific compounds, compositions and methods described herein are specifically described according to certain embodiments , the non-limiting disclosure and reference incorporated herein by reference , the following examples are provided herein. It serves only to illustrate the compounds described and is not intended to be limiting. Each of the references, GenBank
Accession numbers, and the like described herein, are hereby incorporated by reference in their entirety.

The sequence listing attached to this application identifies each sequence as either “RNA” or “DNA” if necessary, but in practice these sequences may be modified by any combination of chemical modifications. Can do. Those skilled in the art will recognize “RNAs for describing modified oligonucleotides.
It is readily understood that such a description as “or“ DNA ”is optional in certain cases, for example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base. , As a DNA with a modified sugar (2′-OH relative to the natural 2′-H of DNA) or as an RNA with a modified base (thymine relative to the natural uracil of RNA (methylated uracil)).

Thus, the nucleic acid sequences presented herein, including but not limited to those in the sequence listing, include (but are not limited to) nucleic acids having modified nucleobases, natural or modified RNA And / or nucleic acids containing any combination of DNA. By way of further example and without limitation, oligomeric compounds having the nucleobase sequence “ATCGATCG” include (but are not limited to) compounds containing RNA bases, whether modified or unmodified. Includes any oligomeric compound having such a nucleobase sequence, including those having the sequence “AUCGAUCG” and some DNA bases such as “AUCGATCG” and some RNA bases. And oligomeric compounds having other modified bases such as “AT ^me CGAUCG”, where ^me C means a cytosine base containing a methyl group at the 5-position.

Example 1-Antisense compound targeting SMN2 The following oligonucleotides were synthesized using standard techniques previously reported.

Example 2-Smn-/-SMN transgenic mice The therapeutic efficacy and safety of the antisense compounds described above can be tested in an appropriate animal model. For example, animal models that appear to be most similar to human disease include those that naturally occur or are induced to develop a particular disease at a high frequency.

In particular, animal models for SMA are known. As mentioned above, the molecular basis of SMA, an autosomal recessive neuromuscular disorder, is homozygous loss of survival motor neuron gene 1 (SMN1). A nearly identical copy of the SMNl gene called SMN2 has been found in humans and modifies the severity of the disease. In contrast to humans, mice have a single gene (Smn) as corresponding to SMN1. The homozygous loss of this gene is lethal to the embryo and results in extensive cell death, meaning that the Smn gene product is required for cell survival and function Yes. Introduction of 2 copies of SMN2 into SMN-deficient mice rescues embryonic lethality and results in mice with the SMA phenotype (Monani et al., Hum. Mol. Genet. (2000) 9 : 333 -339. High copy number SMN2 rescues mice because sufficient SMN protein is produced in motor neurons, as well as Hsieh-Li, which reports the creation of transgenic mouse lines that speak human SMN2. et al., Nat. Genet. (2000) 24 : 66-70 In particular, similar to the spinal cord and skeletal muscle of SMA patients, transgenic mice with SMN2 in the Smn-/-background are spinal cord and The skeletal muscle showed pathological changes, and the severity of the pathological changes in these mice correlated with the amount of SMN protein containing the region encoded by exon 7. Heterogene lacking one copy of Smn Conjugated mice are indicated as Smn-/ + and SMA, type I This is a low-severity model of II.

  The severity of the SMA phenotype is a function of human SMN2 copy number in mice. The “Taiwan” strain has 4 copies of human SMN2, resulting in mice with a moderate to severe SMA phenotype similar to Type I or Type II.

Delta-7 mice (Smn ^{− / −} , hSMN2 ^{+ / +} , SMNΔ7 ^{+ / +} ) also lack mouse Smn and express human SMN2. Delta 7 mice have a more severe phenotype and die soon after birth, typically about 15-20 days after birth.

Example 3-In vivo administration of systemic antisense compounds in Smn-/-SMN2 (Taiwan strain)
Given
Taiwan mice were treated by intraperitoneal injection of saline or 35 mg / kg 396443 or mismatched antisense oligonucleotide control once daily for 5 days and were sacrificed on day 7 two days later. Liver and kidney were harvested and RNA was isolated using standard techniques. SMN2 with exon 7 and SMN2 without exon 7 were visualized by RT-PCR. Administration of ISIS 396443 resulted in a substantial increase in exon 7 content in SMN2 from the kidney and liver compared to saline and mismatch control treated animals.

Example 4-In Vivo Administration of Intracerebroventricular (ICV) Antisense Compound in Smn-/-SMN2 (Taiwan Strain) Taiwan mice were injected with either saline or 150 μg of ISIS 396443 daily by ICV for 7 days . Mice were sacrificed on day 8 and RNA was extracted from the brain and spinal cord. RT-PCR analysis showed a substantial increase in exon 7 content in SMN2 in brain and spinal cord samples obtained from animals treated with ISIS 396443. From these results, exon 7 removal is SMA
Correlation with phenotype indicates that ICV treatment with antisense oligonucleotides targeting SMN can rescue SMA symptoms.

Dose-response Taiwan mice were injected ICV with saline or either 10, 50, 100, or 150 μg of ISIS 396443 daily for 5 days (5 mice for each treatment group) and sacrificed on day 8 It was. RNA was isolated and analyzed by RT-PCR. The 10 μg treatment group showed moderate exon 7 content. The 50 μg, 100 μg, and 150 μg groups all showed substantial exon 7 content.

To determine the duration of response period action, 24 mice were ICV injected daily for 7 days with 50 μg of ISIS 396443. Four mice were sacrificed at the time of the last dose (time 0), and four mice were sacrificed: 1 week, 2 weeks, 4 weeks and 8 weeks after the last dose, respectively. All treated mice showed substantial exon 7 content by RT-PCR with effects at week 8 and no difference from the other groups as shown in FIG.
These results indicate that ISIS 396443 administered ICV at 50 μg per day for 7 days is effective for at least 8 weeks after treatment.

This experiment was repeated and tested for longer periods. Type III mice were treated by ICV infusion of ISIS 396443 at 50 μg / day for 7 days. Mice were sacrificed at 0, 0.5, 1, 2, 4, and 6 months after the end of the 7-day infusion period. RNA was recovered from the spinal cord and analyzed by Northern blot. As shown in the graph below, as shown in FIG. 2, the effect of ISIS 396443 infusion lasted for 6 months after infusion. There are several possible explanations for this long-term effect. It may reflect the stability of ISIS 396443, the modified SMN protein, and / or the dose is very high and the remaining dose will benefit even after the compound is metabolized and lost There is a possibility of continuing. Therefore, these data may support lower doses and occasional doses.

Example 5-Administration of antisense compounds by continuous intracerebroventricular (ICV) infusion Using a micro-osmotic pump (Azlet Osmotic Pumps, Cupertino, CA, USA), ISIS 396443 is adult type with human SMN2 transgene. -III Smn +/- mice or Smn-/-SMA mice (Taiwan strain) were delivered into the cerebrospinal fluid (CSF) via the right ventricle. A dose-response study found that ISIS 396443 compared to about 10% in saline-treated mice.
Intraventricular (ICV) infusion increased the content of SMN2 exon 7 in the spinal cord to approximately 90%.
Western blotting and immunohistochemical analysis showed a robust increase in human transgenic SMN protein levels in spinal motor neurons. These results indicate that the removal of exon 7 is associated with the SMA phenotype and that CNS infusion of antisense oligonucleotide ISIS 396443 can rescue SMA symptoms.

Example 6-Embryo administration
A single ICV injection of either 20 μg or 10 μg of ISIS 396443 was administered to embryonic Taiwan mice on pregnancy day 15 (E15). The animals were sacrificed on postnatal day 7 (P7). RNA was isolated from the lumbar spinal cord and analyzed by RT-PCR. A single embryo administration of ISIS 396443 results in substantial exon 7 inclusions. From these results, since exon 7 removal is associated with the SMA phenotype, in utero treatment with antisense oligonucleotide ISIS 396443,
It is shown that symptoms can be remedied.

The above experiment was repeated and the animals were sacrificed at 11 weeks. Untreated Taiwan mice develop necrotic tails that develop over time. A single embryonic injection of 20 μg of ISIS 396443 significantly delays the onset of tail degradation, as shown in FIG. 3A. These results indicate that embryo treatment with antisense oligonucleotides targeting SMN delays the onset of SMA.

  These results were confirmed in another study using the same conditions, except that the doses tested were 20 μg and 10 μg of ISIS 396443, and that the study included normal mice for comparison. The experimental color results are shown in FIG. 3B.

Example 7-In vivo administration heterozygotes (SMN ^+/- , hSMN2 ^{+ / +} , SMNΔ7 ^{+ / +} ) mating pairs in a Delta-7 mouse model are mated, and on the day of delivery (P0) newborns are , ISIS396443 (18-mer, SEQ ID NO. 1), ISIS396449 (15-mer, SEQ ID NO. 2), ISIS387954 (20-mer, SEQ ID NO. 7) or scrambled control ASO (ISIS439273; 18-mer) ). Mice were injected bilaterally with a total dose of 8 μg (4 μg for each lateral ventricle) into the lateral ventricle. All injections were made using a finely drawn glass pipette needle as described (Passini et al, J. Virol. (2001) 75 : 12382-12392). After injection, the toes of the newborn are cut and genotyped (Le et al., Hum. Mol. Genet. (2005) 14 : 845-857), SMA (SMN ^{− / −} , hSMN2 ^{+ / +} , SMNΔ7 ^{+ / +} ) mice, heterozygous mice, and wild type (SMN ^{+ / +} , hSMN2 ^{+ / +} , SMNΔ7 ^{+ / +} ) mice were identified. All fetuses were removed and 7 newborns served as controls for fetal size for survival. Some fetuses were not injected to generate an untreated control group.

The widely known 18-mer was detected in the spinal cord including the thoracic, lumbar, and cervical regions of the spinal cord 14 days after injection in SMA mice. Furthermore, colocalization studies with ChAT confirmed that the majority of cells targeted by ISIS 396443 in the spinal cord were motor neurons. No signal was detected in control and untreated mice.

Western blot analysis at 14 days showed the amount of SMN in the brain and spinal cord, which was 40-60% of the wild type level compared to 10% for the untreated SMA control. No signal on the background was detected in control mice treated with a scrambled version of ASO. Western blot results are presented in FIG.

Regardless of ASO length, SMA mice treated with SMA ASO, compared to untreated SMA mice or SMA mice treated with scrambled ASO, showed weight, gait function (righting reflex and grip strength), and coordination ( A significant increase in hindlimb limbs) was observed. No significant increase in body weight, gait function (righting reflex and grip strength), or coordination was found in SMA mice treated with scrambled ASO compared to untreated SMA mice. The results are provided in FIGS. 5 and 6.

Importantly, as shown in FIG. 7, regardless of the length of ASO, SMA mice treated with ASO produce a significant increase in median survival. Survival was calculated from birth and was 31.5 (15-mer), 27.0 (18-mer), and 28.0 (20-mer) compared to 16.0 for untreated SMA controls. In contrast, SMA mice treated with 18-mer scrambled controls improved survival. These results indicated that treatment with antisense oligonucleotides targeting SMN treatment increased the lifespan of subjects with SMA.

  SMA ASO also increases the number of motor neuron cells in the spinal cord, as shown in FIG.

SMN RNA was measured by RT-PCR. Compared to untreated SMA mice, animals treated with SMA ASO increased SMN RNA levels. The results obtained from mice treated with 20-mer ASO as compared to untreated SMA mice are shown in FIG.

To examine whether survival is further increased by administration of the second dose, the above experiment was repeated on day 21 with an additional dose of 20 μg. The results are shown in FIG. The graph above shows the effect of the first dose of 8 μg on day 0. On day P21, half of the treated mice were treated a second time.

  The effect of the second treatment is shown in FIG. 11 compared to the mice that received only the first treatment. This result suggests that a second ICV treatment with antisense oligonucleotide further increases survival.

Example 8-Activity in SMA type III mice
Two antisense compounds and one control compound were tested in a mouse model of SMA. The compounds are listed in the table below.

The Taiwan line of SMA type III mice was obtained from The Jackson Laboratory (Bar Harbor, Maine). These mice are deficient in murine SMN and are homozygous for human SMN2 (mSMN − / −; hSMN2 + / +). These mice were described below; Hsieh-Li HM, et al., Nature Genet. 24, 66-70 2000.

Mice were treated with 3, 10, 30, or 100 μg ISIS 396443 or ISIS 449220 per day or with 30 or 100 μg control compound ISIS 439272 per day in phosphate buffered saline (PBS). Control mice were treated with PBS only (0 dose). All treatments, Azlet 1007D
Administration was by intracerebroventricular (ICV) infusion using an osmotic pump. However,
There were 5 animals for each dose, and 2 of the mice from the highest dose of ISIS 449220 died before the end of the study. The animals were sacrificed on day 9 (2 days after the last dose) and spinal cord brain and lumbar sections were taken from each animal. Real-time PCR was performed on each sample to determine the amount of human SMN2 message with exon 7 ((+) exon 7) and the amount of human SMN2 message without exon 7 ((−) exon 7). Real-time PCR was similarly performed to determine the expression levels of allograft inflammatory factor (AIF1) and glyceraldehyde 3-phosphate dehydrogenase (GADPH).

Expression levels for (+) exon 7 and (−) exon 7 were normalized to GADPH levels. The normalized expression level was divided by the GADPH-normalized level obtained from PBS treated control mice. The magnification-control values obtained are shown in Table 17 below. The data shows the mean fold of the control of all 5 mice in each group except for the highest dose of ISIS 449220, which shows that there are 3 surviving mice.

Administration of ISIS 396443 resulted in a significant increase in exon 7 capsules. 10 μg /
In the day, ISIS 396443 resulted in an SMN2 message in the brain that resulted in nearly twice as much (1.8 times) remaining exon 7, and more than doubled in the lumbar spinal cord when compared to untreated controls .

The expression of allograft inflammatory factor (AIF1) was tested as a measure of inflammation. After all samples were normalized to (GADPH), the ratio of AIF1 for each treatment group was divided by the value for the PBS control. ISIS 396443 did not result in an increase in AIF1, even at higher doses. ISIS449220 produced an increase in AIF1 in both the brain and lumbar spinal cord. The data in Table 18 shows the mean fold of the control for all 5 mice in each group except for the highest dose of ISIS 449220, where 3 surviving mice are shown.

Example 9-Administration to monkeys Cynomolgus was used to evaluate the distribution of ISIS 395443 for different doses and different routes of administration. ISIS 396443 was administered to 2 monkeys. One monkey received 3 mg by ICV infusion, and the other monkey received 3 mg by IT infusion. Both infusions were delivered over a 24 hour period. These monkeys were sacrificed 96 hours after the end of the infusion period and the tissues were collected. The concentration of ISIS 396443 was measured in samples from the cervical, thoracic and lumbar portions of the spinal cord. The results are summarized in the following table.

   Since cynololgus is approximately 3 kg, this dose is approximately 1 mg / kg.

  To further evaluate the distribution of ISIS 39644, 26 monkeys were divided into 6 groups as shown in the table below.

The infusion rate was 100 μL / hour for all groups. Group 1 received all 3 mg of ISIS39644 in saline, except that only saline was administered.
Monkeys were sacrificed 5 days after the end of the infusion and tissues were collected.

The concentration of ISIS 39644 in monkey-derived tissue samples was assessed using standard techniques.
A summary of the results is provided in the graph of FIG.

  Samples were also evaluated histologically. Histology showed no adverse effects of treatment and confirmed the presence of ISIS 396443. There was no evidence of Purkinje cell loss.

The rapid infusion seemed to have more ISIS 396443 compared to the slower infusion. These results suggest that faster infusion rates or bolus injection may be preferred in certain embodiments. Because bolus administration has certain practical advantages over infusion, in certain embodiments, it is the preferred method of administration into CSF. In certain embodiments, the preferred method of administration into CSF is by bolus IT injection.

Example 10- Generation of a mouse model of severe SMA and ICV treatment Mice with a severe SMA phenotype (sSMA mice) were produced. Homozygous sSMA mice have 2 copies of human SMN2 and no mouse SMN. The average life span is about 10 days. In addition, SMA mice have a smaller and shorter tail. Heterozygotes have mouse SMN and develop normally.

To study the effects of antisense compounds in these sSMA mice, 20 μg of ISIS 396443 was injected into ICV on day P1. Treatment results in an average survival of 9.9 days (saline-treated control)
It increased from 16.7 days. Full-length SMN RNA in tissues from treated mice by RT-PCR analysis
Increased.

Example 11-Systemic administration of ISIS 396443
sSMA mice and healthy heterozygous control mice were grouped and the effect of ISIS 396443 was studied by bolus ICV injection and / or bolus subcutaneous injection (SC) as follows:
Group 1 -ICV + SC
Deliver 20 μg on P1 or P2 for a single ICV injection (day 1 or 2 after birth); and 50 μg / g between P0 and P3 for two subcutaneous injections.

Group 2 -SC + SC
Two SC injections deliver 50 μg / g between P0 and P3; and one subcutaneous injection delivers 50 μg / g between P5 and P6; and 50 μg / g P9 for subcutaneous injection And delivered between P10.

Group 3 -SC
Two SC injections deliver 50 μg / g between P0 and P3.

Group 4 -SMA saline control
For one ICV injection, saline is delivered to P1 or P2; and for two subcutaneous injections, saline is delivered between P0 and P3.

Group 5 -heterozygous control
For one ICV injection, 20 μg is delivered to P1 or P2; and for two subcutaneous injections, 50 μg / g is delivered between P0 and P3 in heterozygous mice.

  Each group contained 14-22 mice. Survival (days) for individual mice in each group is presented in the table below. Many mice in this study are still alive when preparing the patent application. Thus, a value with a “>” indicates that the mouse has survived that day and is still alive.

Example 12-SC Administration Dose-Response The survival of sSMA mice that received different doses of subcutaneous ISIS 396443 was evaluated by the following administration groups.

Group 1- SC400 (dose range 80 mg / kg to 180 mg / kg)
Two SC injections, 400 μg per mouse in total, delivered to P0-P3, the first dose is 150 μg (3 μl volume) on P0 or P1, and the second dose is 250 μg delivered to P2 or P3 Was (5 μl volume).

Group 2- SC200 (40 mg / kg to 90 mg / kg dose range)
Two SC injections, delivering a total volume of 200 μg per mouse between P0 and P3, the first dose is 75 μg (1.5 μl volume) from P0 to P1, and the second dose is to P2 or P3 125 μg delivered (2.5 μl volume).

Group 3- SC100 (dose range of 20 mg / kg to 45 mg / kg)
2 SC injections, 100 μg per mouse in total delivered between P0 and P3, 1st dose delivered to P0 or P1 40 μg (2 μl volume) and 2nd dose delivered to P2 or P3 Was 60 μg (3 μl volume).

Group 4-SMA saline (negative control)
2 SC injections of saline between P0 and P3, 1st injection was in P0 or P1 (5 μl volume), and 2nd injection was delivered to P2 or P3 (5 μl volume) .

Group 5-heterozygous control (positive control)
Mice without treatment.

  Each group contained 14 to 26 mice. Survival (days) for individual mice in each group is presented in the table below. Many mice in this study are still alive when preparing the patent application. Thus, a value with a “>” indicates that the mouse has survived that day and is still alive.

Example 13-ICV Infusion vs. Administration by ICV Bolus Intraventricular Bolus Infusion (ICV Bolus) was compared to administration by continuous intracerebroventricular infusion (ICV infusion). ISIS387954 was administered to SMA type III transgenic mice. ICV-infused mice were injected with a total volume of 0 (PBS control), 87.5 μg, 175 μg, 350 μg, or 700 μg over 7 days and then sacrificed 2 days later. For ICV bolus mice
The same total volume, 0 (PBS control), 87.5 μg, 175 μg, 350 μg, or 700 μg was injected with a single ICV injection and then sacrificed 9 days later. There were 5 mice in each group. RNA was collected from the lumbar spinal cord and analyzed by real-time PCR. Intron 7 content was normalized to saline-treated controls. The results are summarized in the following table.

In this experiment, the same dose as delivered by ICV bolus infusion resulted in ICV
Higher activity was obtained than when delivered by infusion over 7 days.

Real-time PCR was also performed to measure inflammation by measuring the expression level of allograft inflammatory factor (AIF1). None of the samples from the treated mice showed significant differences from the control mice.

Example 14-Dose with ICV bolus-Response Intraventricular bolus administration was tested at additional doses. Transgenic mice were administered 0, 10.9 μg, 21.9 μg, 43.4 μg, 87.5 μg, or 175 μg of ISIS387954 by a single bolus ICV injection and sacrificed after 9 days, as shown in Example 13. I was killed. Samples were taken from the brain and lumbar spinal cord. RNA was prepared and analyzed by RT-PCR for changes in intron 7 content and for changes in AIF1. None of the samples showed changes in AIF1 compared to the control. The results from intron 7 inclusions are summarized in the following table. The ED50 is approximately 22 μg.

Claims (107)

  A method comprising administering to a subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 pre-mRNA, wherein the antisense compound is cerebrospinal fluid Said method being administered in.   2. The method of claim 1, wherein administration is in the subarachnoid space.   2. The method of claim 1, wherein the administration is performed in cerebrospinal fluid within the brain.   4. The method according to any of claims 1-3, wherein administering comprises bolus injection.   4. The method according to any of claims 1-3, wherein the administration comprises infusion using a delivery pump.   6. The method of any of claims 1-5, wherein the antisense compound is administered at a dose of 0.01-10 milligrams of antisense compound per kg body weight of the subject.   7. The method of claim 6, wherein the dose is 0.01 to 10 milligrams of antisense compound per kg body weight of the subject. 7. The method of claim 6, wherein the dose is 0.01 to 5 milligrams of antisense compound per kg body weight of the subject. 7. The method of claim 6, wherein the dose is 0.05 to 1 milligram of antisense compound per kg body weight of the subject. 7. The method of claim 6, wherein the dose is 0.01 to 0.5 milligrams of antisense compound per kg body weight of the subject. 7. The method of claim 6, wherein the dose is 0.05 to 0.5 milligrams of antisense compound per kg body weight of the subject.   12. The method according to any of claims 6-11, wherein the dose is administered daily.   12. The method according to any of claims 6 to 11, wherein the dose is administered once a week. 12. The method according to any of claims 6 to 11, wherein the antisense compound is administered continuously and the dose is the amount administered per day. 14. A method according to any of claims 1 to 13, comprising administering at least one induction dose during the induction phase and administering at least one maintenance dose during the maintenance phase. The induction phase is 0.05 to 5.0 milligrams of antisense compound per kg body weight of the subject,
The method of claim 15. 17. The method of claim 15 or 16, wherein the maintenance dose is 0.01 to 1.0 milligram of antisense compound per kg body weight of the subject. 18. A method according to any of claims 15 to 17, wherein the duration of the induction phase is at least 1 week. 19. A method according to any of claims 15-18, wherein the duration of the maintenance phase is at least 1 week. 20. A method according to any of claims 15-19, wherein each induction administration and each maintenance administration comprises a single injection. 20. A method according to any of claims 15-19, wherein each induction administration and each maintenance administration independently comprises two or more injections.   24. The method of any of claims 1-21, wherein the antisense compound is administered at least twice over a treatment period of at least 1 week.   24. The method of claim 22, wherein the treatment period is at least 1 month.   24. The method of claim 22, wherein the treatment period is at least 2 months.   24. The method of claim 22, wherein the treatment period is at least 4 months. 26. The method of any of claims 15-25, wherein the induction phase is administered by one or more bolus infusions and the maintenance dose is administered by an infusion pump.   27. A method according to any of claims 1 to 26 comprising assessing the tolerability and / or efficacy of the antisense compound.   28. A method according to any of claims 1 to 27, wherein the dosage or frequency of administration of the antisense compound is reduced according to an indication that administration of the antisense compound is not acceptable. 29. The method of any of claims 1-28, wherein the dosage or frequency of administration of the antisense compound is maintained or reduced according to the indication for which administration of the antisense compound is effective.   30. The method of any of claims 1-29, wherein the dosage of the antisense compound is increased according to an indication for which administration of the antisense compound is not effective.   31. A method according to any of claims 1 to 30, wherein the frequency of administration of the antisense compound is reduced according to the indication for which administration of the antisense compound is effective.   32. The method of any of claims 1-31, wherein the frequency of administration of the antisense compound is increased according to an indication for which administration of the antisense compound is not effective. 33. The method of any of claims 1-32, comprising co-administering an antisense compound and at least one other treatment.   34. The method of claim 33, wherein the antisense compound and at least one other treatment are co-administered simultaneously. 35. The method of claim 34, wherein the antisense compound is administered prior to administration of at least one other treatment.   35. The method of claim 34, wherein the antisense compound is administered after administration of at least one other treatment. 37. The method of any of claims 33-36, wherein the at least one other treatment comprises administering one or more of valproic acid, riluzole, hydroxyurea, and butyrate. 34. The at least one other treatment comprises administering trichostatin-A.
A method according to any one of -37. 39. The method of any of claims 33-38, wherein the at least one other treatment comprises administering a stem cell. 40. A method according to any of claims 33 to 39, wherein the at least one other treatment is gene therapy. About 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 50 mg of antisense compound / ml, or at a concentration of about 100 mg / ml,
41. A method according to any one of claims 1-40. 2. Increasing the content of exon 7 of SMN2 mRNA in a motor neuron of a subject.
A method according to any one of -41. 43. The method of any one of claims 1-42, wherein the content of exon 7 amino acids of the SMN2 polypeptide in the subject's motor neurons is increased. Administering to the subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2, and thereby, in the subject's motor neurons, of SMN2 mRNA Increasing the content of exon 7 of the SMN2 mRNA in a motor neuron of the subject, comprising increasing the content of exon 7. Administering to a subject an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2, and thereby in an SMN2 polypeptide in the subject's motor neurons Increasing the amino acid content of exon 7 in the SMN2 polypeptide in a motor neuron of the subject, comprising increasing the content of exon 7 amino acid.   46. The method according to any of claims 1-45, wherein the subject has SMA.   46. The method according to any of claims 1-45, wherein the subject has type I SMA.   46. The method according to any of claims 1-45, wherein the subject has type II SMA.   46. The method of any of claims 1-45, wherein the subject has type III SMA.   50. The method of any of claims 1-49, wherein the first dose is administered into the uterus. 51. The method of claim 50, wherein the first dose is administered prior to completing the formation of blood brain barrier formation. 50. The method of any one of claims 1-49, wherein the first dose is administered within one week of life of the subject. 50. The method of any of claims 1-49, wherein the first dose is administered within one month of life of the subject. 50. The method of any one of claims 1-49, wherein the first dose is administered within 3 months of life of the subject. 50. The method of any one of claims 1-49, wherein the first dose is administered within 6 months of life of the subject.   50. The method of any of claims 1-49, wherein the first dose is administered when the subject is 1-2 years old. 50. The method of any of claims 1-49, wherein the first dose is administered when the subject is 1-15 years old.   50. The method of any of claims 1-49, wherein the first dose is administered when the subject is 15 years of age or older.   59. The method according to any of claims 1 to 58, wherein the subject is a mammal.   60. The method of claim 59, wherein the subject is a human.   61. A method according to any of claims 1-60, comprising identifying a subject having SMA. Identifying a subject by measuring the electrical activity of one or more subject's muscles;
62. The method of claim 61.   62. The method of claim 61, wherein the subject is identified by genetic testing to determine whether the subject has a mutation in the subject's SMN1 gene.   62. The method of claim 61, wherein the subject is identified by muscle biopsy.   43. The method of claim 42, wherein administering an antisense compound results in an increase in the amount of SMN2 mRNA having at least 10% exon 7.   66. The method of claim 65, wherein the increase in the amount of SMN2 mRNA having exon 7 is at least 20%.   66. The method of claim 65, wherein the increase in the amount of SMN2 mRNA having exon 7 is at least 50%.   66. The method of claim 65, wherein the increase in the amount of SMN2 mRNA having exon 7 is at least 70%. SMN with at least 10% exon 7 amino acids by administration of antisense compound
44. The method of claim 43, resulting in an increase in the amount of 2 polypeptides. 70. The method of claim 69, wherein the increase in the amount of SMN2 polypeptide having an amino acid of exon 7 is at least 20%.   70. The method of claim 69, wherein the increase in the amount of SMN2 polypeptide having an amino acid of exon 7 is at least 50%.   70. The method of claim 69, wherein the increase in the amount of SMN2 polypeptide having an amino acid of exon 7 is at least 70%.   75. The method of any of claims 1-72, wherein administering an antisense compound ameliorates at least one symptom of SMA in the subject.   74. The method of claim 73, wherein administering an antisense compound results in an improvement of the subject's motor function.   74. The method of claim 73, wherein administering an antisense compound results in delaying or reducing loss of motor function in the subject.   75. The method of claim 73, wherein administering an antisense compound results in improved respiratory function.   75. The method of claim 73, wherein administering an antisense compound results in improved survival. 78. The method of any of claims 1-77, wherein at least one nucleoside of the antisense oligonucleotide comprises a modified sugar moiety.   80. The method of claim 78, wherein the at least one modified sugar component comprises a 2'-methoxyethyl sugar component.   80. The method of any of claims 1-79, wherein essentially each nucleoside of the antisense oligonucleotide comprises a modified sugar moiety. 81. The method of claim 80, wherein all nucleosides comprising a modified sugar moiety contain the same sugar modification. 84. The method of claim 81, wherein each modified sugar component comprises a 2'-methoxyethyl sugar component.   80. The method of any of claims 1-79, wherein each nucleoside of the antisense oligonucleotide comprises a modified sugar component.   84. The method of claim 83, wherein all of the nucleosides contain the same sugar modification. 85. The method of claim 84, wherein each modified sugar component comprises a 2'-methoxyethyl sugar component. 86. The method of any of claims 1-85, wherein the at least one internucleoside linkage is a phosphorothioate internucleoside linkage.   90. The method of claim 86, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.   90. The method of any of claims 1-87, wherein the antisense oligonucleotide consists of 10-25 linked nucleosides.   90. The method of any of claims 1-87, wherein the antisense oligonucleotide consists of 12-22 linked nucleosides.   90. The method of any of claims 1-87, wherein the antisense oligonucleotide consists of 15-20 linked nucleosides. The antisense oligonucleotide consists of 18 linked nucleosides.
A method according to any one of -87.   92. The method according to any of claims 1 to 91, wherein the antisense oligonucleotide is at least 90% complementary to a nucleic acid encoding human SMN2.   94. The method of claim 92, wherein the antisense oligonucleotide is completely complementary to a nucleic acid encoding human SMN2.   94. The method according to any of claims 1 to 93, wherein the oligonucleotide has a nucleobase sequence comprising at least 10 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 1.   95. The method of claim 94, wherein the oligonucleotide has a nucleobase sequence comprising at least 15 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 1.   95. The method of claim 94, wherein the oligonucleotide has a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1.   95. The method of claim 94, wherein the oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence SEQ ID NO: 1. 98. The method according to any of claims 1 to 97, wherein the antisense compound comprises a conjugated group or a terminal group. 98. The method according to any one of claims 1 to 97, wherein the antisense compound consists of an antisense oligonucleotide. 100. The method of any of claims 1-99, wherein the antisense compound is also administered systemically. 101. The method of claim 100, wherein the systemic administration is by intravenous injection or intraperitoneal injection.   102. The method of claim 100 or 101, wherein systemic administration and administration to the central nervous system are performed simultaneously.   102. The method of claim 100 or 101, wherein the systemic administration and administration to the central nervous system are performed at different times. 104. An antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 for use in the method of any one of claims 1-103. 105. The antisense compound of claim 104 for use in treating a disease or condition associated with survival motor neuron 1 (SMN1). Use of an antisense compound comprising an antisense oligonucleotide complementary to intron 7 of a nucleic acid encoding human SMN2 in the manufacture of a medicament for use in the method of any one of claims 1-105. . 107. Use according to claim 106, wherein the medicament is for treating a disease or condition associated with survival motor neuron 1 (SMN1).

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